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Overview of the 
Immune System 



7 HE IMMUNE SYSTEM IS A REMARKABLY VERSATILE 

defense system that has evolved to protect animals 
from invading pathogenic microorganisms and 
cancer. It is able to generate an enormous variety of cells and 
molecules capable of specifically recognizing and eliminat- 
ing an apparently limitless variety of foreign invaders. These 
cells and molecules act together in a dynamic network whose 
complexity rivals that of the nervous system. 

Functionally, an immune response can be divided into 
two related activities — recognition and response. Immune 
recognition is remarkable for its specificity. The immune 
system is able to recognize subtle chemical differences that 
distinguish one foreign pathogen from another. Further- 
more, the system is able to discriminate between foreign 
molecules and the body's own cells and proteins. Once a for- 
eign organism has been recognized, the immune system 
recruits a variety of cells and molecules to mount an appro- 
priate response, called an effector response, to eliminate or 
neutralize the organism. In this way the system is able to 
convert the initial recognition event into a variety of effector 
responses, each uniquely suited for eliminating a particular 
type of pathogen. Later exposure to the same foreign organ- 
ism induces a memory response, characterized by a more 
rapid and heightened immune reaction that serves to elimi- 
nate the pathogen and prevent disease. 

This chapter introduces the study of immunology from 
an historical perspective and presents a broad overview of 
the cells and molecules that compose the immune system, 
along with the mechanisms they use to protect the body 
against foreign invaders. Evidence for the presence of very 
simple immune systems in certain invertebrate organisms 
then gives an evolutionary perspective on the mammalian 
immune system, which is the major subject of this book. El- 
ements of the primitive immune system persist in verte- 
brates as innate immunity along with a more highly evolved 
system of specific responses termed adaptive immunity. 
These two systems work in concert to provide a high degree 
of protection for vertebrate species. Finally, in some circum- 
stances, the immune system fails to act as protector because 
of some deficiency in its components; at other times, it be- 
comes an aggressor and turns its awesome powers against its 
own host. In this introductory chapter, our description of 
immunity is simplified to reveal the essential structures and 
function of the immune system. Substantive discussions, ex- 
perimental approaches, and in-depth definitions are left to 
the chapters that follow. 




■ Historical Perspective 

■ Innate Immunity 

■ Adaptive Immunity 

■ Comparative Immunity 

■ Immune Dysfunction and Its Consequences 



Like the later chapters covering basic topics in immu- 
nology, this one includes a section called "Clinical Focus" 
that describes human disease and its relation to immunity. 
These sections investigate the causes, consequences, or treat- 
ments of diseases rooted in impaired or hyperactive immune 
function. 



Historical Perspective 

The discipline of immunology grew out of the observation 
that individuals who had recovered from certain infectious 
diseases were thereafter protected from the disease. The 
Latin term immunis, meaning "exempt," is the source of the 
English word immunity, meaning the state of protection 
from infectious disease. 

Perhaps the earliest written reference to the phenomenon 
of immunity can be traced back to Thucydides, the great his- 
torian of the Peloponnesian War. In describing a plague in 
Athens, he wrote in 430 bc that only those who had recov- 
ered from the plague could nurse the sick because they 
would not contract the disease a second time. Although early 
societies recognized the phenomenon of immunity, almost 



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two thousand years passed before the concept was success- 
fully converted into medically effective practice. 

The first recorded attempts to induce immunity deliber- 
ately were performed by the Chinese and Turks in the fif- 
teenth century. Various reports suggest that the dried crusts 
derived from smallpox pustules were either inhaled into the 
nostrils or inserted into small cuts in the skin (a technique 
called variolation). In 1718, Lady Mary Wortley Montagu, the 
wife of the British ambassador to Constantinople, observed 
the positive effects of variolation on the native population 
and had the technique performed on her own children. The 
method was significantly improved by the English physician 
Edward Jenner, in 1798. Intrigued by the fact that milkmaids 
who had contracted the mild disease cowpox were subse- 
quently immune to smallpox, which is a disfiguring and of- 
ten fatal disease, Jenner reasoned that introducing fluid from 
a cowpox pustule into people (i.e., inoculating them) might 
protect them from smallpox. To test this idea, he inoculated 
an eight-year-old boy with fluid from a cowpox pustule and 
later intentionally infected the child with smallpox. As pre- 
dicted, the child did not develop smallpox. 

Jenner's technique of inoculating with cowpox to protect 
against smallpox spread quickly throughout Europe. How- 
ever, for many reasons, including a lack of obvious disease 
targets and knowledge of their causes, it was nearly a hun- 
dred years before this technique was applied to other dis- 
eases. As so often happens in science, serendipity in 
combination with astute observation led to the next major 
advance in immunology, the induction of immunity to 
cholera. Louis Pasteur had succeeded in growing the bac- 
terium thought to cause fowl cholera in culture and then had 
shown that chickens injected with the cultured bacterium de- 
veloped cholera. After returning from a summer vacation, he 
injected some chickens with an old culture. The chickens be- 
came ill, but, to Pasteur's surprise, they recovered. Pasteur 
then grew a fresh culture of the bacterium with the intention 
of injecting it into some fresh chickens. But, as the story goes, 
his supply of chickens was limited, and therefore he used the 
previously injected chickens. Again to his surprise, the chick- 
ens were completely protected from the disease. Pasteur 
hypothesized and proved that aging had weakened the viru- 
lence of the pathogen and that such an attenuated strain 
might be administered to protect against the disease. He 
called this attenuated strain a vaccine (from the Latin vacca, 
meaning "cow"), in honor of Jenner's work with cowpox 

Pasteur extended these findings to other diseas 
strating that it was possible to attenuate, or 
pathogen and administer the attenuated strain a 
In a now classic experiment at Pouilly-le-Fort ii 
teur first vaccinated one group of sheep with heat-attenuated 
anthrax bacillus {Bacillus anthracis); he then challenged the 
vaccinated sheep and some unvaccinated sheep with a viru- 
lent culture of the bacillus. All the vaccinated sheep lived, and 
all the unvaccinated animals died. These experiments 
marked the beginnings of the discipline of immunology. In 



ss, demon- 



l 1881, Pas- 




Wood engraving of Loi 
Meister receive the rabies vaccine. [From H 
courtesy of the National Library of Medicine.] 



hing Joseph 
Weekly 29:836; 



1885, Pasteur administered his first vaccine to a human, a 
young boy who had been bitten repeatedly by a rabid dog 
(Figure 1-1). The boy, Joseph Meister, was inoculated with a 
series of attenuated rabies virus preparations. He lived and 
later became a custodian at the Pasteur Institute. 

Early Studies Revealed Humoral and Cellular 
Components of the Immune System 

Although Pasteur proved that vaccination worked, he did not 
understand how. The experimental work of Emil von 
Behring and Shibasaburo Kitasato in 1890 gave the first in- 
sights into the mechanism of immunity, earning von Behring 
the Nobel prize in medicine in 1901 (Table 1-1). Von Behring 
and Kitasato demonstrated that serum (the liquid, noncellu- 
lar component of coagulated blood) from animals previously 
immunized to diphtheria could transfer the immune state to 
unimmunized animals. In search of the protective agent, var- 
ious researchers during the next decade demonstrated that 
an active component from immune serum could neutralize 
toxins, precipitate toxins, and agglutinate (clump) bacteria. 
In each case, the active agent was named for the activity it ex- 
hibited: antitoxin, precipitin, and agglutinin, respectively. 



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| TABLE 1- 
Year 


Recipient 


Country 


Research 


1901 


EmilvonBehring 


Germany 


Serum antitoxins 


1905 


Robert Koch 


Germany 


Cellular immunity to tuberculosis 


1908 


Elie MetchnikofF 

Paul Ehrlich 


Russia 
Germany 


Role of phagocytosis (MetchnikofF) and 
antitoxins (Ehrlich) in immunity 


1913 


Charles Richet 


France 


Anaphylaxis 


1919 


Jules Border 


Belgium 


Complement-mediated bacteriolysis 


1930 


Karl Landsteiner 


United States 


Discovery of human blood groups 


1951 


MaxTheiler 


South Africa 


Development of yellow fever vaccine 


1957 


Daniel Bovet 


Switzerland 


Antihistamines 


1960 


F. Macfarlane Burnet 
Peter Medawar 


Australia 
Great Britain 


Discovery of acquired immunological 
tolerance 


1972 


Rodney R. Porter 
Gerald M. Edelman 


Great Britain 
United States 


Chemical structure of antibodies 


1977 


Rosalyn R. Yalow 


United States 


Development of radioimmunoassay 


1980 


George Snell 
Jean Daussct 
Baruj Benacerraf 


United States 


Major histocompatibility complex 




United States 




1984 


Cesar Milstein 
Georges E. Kohler 


Great Britain 
Germany 


Monoclonal antibody 




Niels K.Jerne 


Denmark 


Immune regulatory theories 


1987 


Susumu Tonegawa 


Japan 


Gene rearrangement in antibody 


1991 


E. Donnall Thomas 
Joseph Murray 


United States 
United States 


Transplantation immunology 


1996 


Peter C. Doherty 
Rolf M. Zinkernagel 


Australia 
Switzerland 


Role of major histocompatibility complex 
in antigen recognition by by T cells 



Initially, a different serum component was thought to be re- 
sponsible for each activity, but during the 1930s, mainly 
through the efforts of Elvin Kabat, a fraction of serum first 
called gamma-globulin (now immunoglobulin) was shown 
to be responsible for all these activities. The active molecules 
in the immunoglobulin fraction are called antibodies. Be- 
cause immunity was mediated by antibodies contained in 
body fluids (known at the time as humors), it was called hu- 
moral immunity. 

In 1883, even before the discovery that a serum compo- 
nent could transfer immunity, Elie Metchnikoff demon- 
strated that cells also contribute to the immune state of an 
animal. He observed that certain white blood cells, which he 
termed phagocytes, were able to ingest (phagocytose) mi- 
croorganisms and other foreign material. Noting that these 
phagocytic cells were more active in animals that had been 
immunized, Metchnikoff hypothesized that cells, rather than 
serum components, were the major effector of immunity. 
The active phagocytic cells identified by Metchnikoff were 
likely blood monocytes and neutrophils (see Chapter 2). 



In due course, a controversy developed between those 
who held to the concept of humoral immunity and those 
who agreed with Metchnikoff 's concept of cell-mediated im- 
munity. It was later shown that both are correct — immunity 
requires both cellular and humoral responses. It was difficult 
to study the activities of immune cells before the develop- 
ment of modern tissue culture techniques, whereas studies 
with serum took advantage of the ready availability of blood 
and established biochemical techniques. Because of these 
technical problems, information about cellular immunity 
lagged behind findings that concerned humoral immunity. 

In a key experiment in the 1940s, Merrill Chase succeeded 
in transferring immunity against the tuberculosis organism 
by transferring white blood cells between guinea pigs. This 
demonstration helped to rekindle interest in cellular immu- 
nity. With the emergence of improved cell culture techniques 
in the 1950s, the lymphocyte was identified as the cell re- 
sponsible for both cellular and humoral immunity. Soon 
thereafter, experiments with chickens pioneered by Bruce 
Glick at Mississippi State University indicated that there were 



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two types of lymphocytes: T lymphocytes derived from the 
thymus mediated cellular immunity, and B lymphocytes 
from the bursa of Fabricius (an outgrowth of the cloaca in 
birds) were involved in humoral immunity. The controversy 
about the roles of humoral and cellular immunity was re- 
solved when the two systems were shown to be intertwined, 
and that both systems were necessary for the immune 
response. 

Early Theories Attempted to Explain 
the Specificity of the Antibody- 
Antigen Interaction 

One of the greatest enigmas facing early immunologists was 
the specificity of the antibody molecule for foreign material, 
or antigen (the general term for a substance that binds with 
a specific antibody). Around 1900, Jules Bordet at the Pasteur 
Institute expanded the concept of immunity by demonstrat- 
ing specific immune reactivity to nonpathogenic substances, 
such as red blood cells from other species. Serum from an an- 
imal inoculated previously with material that did not cause 
infection would react with this material in a specific manner, 
and this reactivity could be passed to other animals by trans- 
ferring serum from the first. The work of Karl Landsteiner 
and those who followed him showed that injecting an animal 
with almost any organic chemical could induce production 
of antibodies that would bind specifically to the chemical. 
These studies demonstrated that antibodies have a capacity 
for an almost unlimited range of reactivity, including re- 
sponses to compounds that had only recently been synthe- 
sized in the laboratory and had not previously existed in 
nature. In addition, it was shown that molecules differing in 
the smallest detail could be distinguished by their reactivity 
with different antibodies. Two major theories were proposed 
to account for this specificity: the selective theory and the in- 
structional theory. 

The earliest conception of the selective theory dates to Paul 
Ehrlich in 1900. In an attempt to explain the origin of serum 
antibody, Ehrlich proposed that cells in the blood expressed a 
variety of receptors, which he called "side-chain receptors," 
that could react with infectious agents and inactivate them. 
Borrowing a concept used by Emil Fischer in 1894 to explain 
reen an enzyme and its substrate, Ehrlich 
ing of the receptor to an infectious agent 
een a lock and key. Ehrlich suggested that 
l an infectious agent and a cell-bound 
e the cell to produce and release more 
me specificity. According to Ehrlich's 
theory, the specificity of the receptor was determined before 
its exposure to antigen, and the antigen selected the appro- 
>r. Ultimately all aspects of Ehrlich's theory 
/en correct with the minor exception that the 
;ts as both a soluble antibody molecule and as a 
:eptor; it is the soluble form that is secreted 



'oposed that bin 
as like the fit bet 



■ would indui 
•s with the si 









cell-bound r 



rather than the bound form released. 



In the 1930s and 1940s, the selective theory was chal- 
lenged by various ins ries, in which antigen 
played a central role in determining the specificity of the an- 
tibody molecule. According to the instructional theories, a 
particular antigen would serve as a template around which 
antibody would fold. The antibody molecule would thereby 
assume a configuration complementary to that of the antigen 
template. This concept was first postulated by Friedrich 
Breinl and Felix Haurowitz about 1930 and redefined in the 
1940s in terms of protein folding by Linus Pauling. The in- 
structional theories were formally disproved in the 1960s, by 
which time information was emerging about the structure of 
DNA, RNA, and protein that would offer new insights into 
the vexing problem of how an individual could make anti- 
bodies against almost anything. 

In the 1950s, selective theories resurfaced as a result of 
new experimental data and, through the insights of Niels 
Jerne, David Talmadge, and F. Macfarlane Burnet, were re- 
fined into a theory that came to be known as the clonal- 
selection theory. According to this theory, an individual 
lymphocyte expresses membrane receptors that are specific 
for a distinct antigen. This unique receptor specificity is de- 
termined before the lymphocyte is exposed to the antigen. 
Binding of antigen to its specific receptor activates the cell, 
causing it to proliferate into a clone of cells that have the 
same immunologic specificity as the parent cell. The clonal- 
selection theory has been further refined and is now accepted 
as the underlying paradigm of modern immunology. 

The Immune System Includes Innate and 
Adaptive Components 

Immunity — the state of protection from infectious disease 
— has both a less specific and more specific component. The 
less specific component, innate immunity, provides the first 
line of defense against infection. Most components of innate 
immunity are present before the onset of infection and con- 
stitute a set of disease-resistance mechanisms that are not 
specific to a particular pathogen but that include cellular and 
molecular components that recognize classes of molecules 
peculiar to frequently encountered pathogens. Phagocytic 
cells, such as macrophages and neutrophils, barriers such as 
skin, and a variety of antimicrobial compounds synthesized 
by the host all play important roles in innate immunity. In 
contrast to the broad reactivity of the ii 
tern, which is uniform in 
cific component, adaptr 
play until there is an antigen; 
Adaptive immunity responds ti 
gree of specificity as well as 
"memory." Typically, there is a 
against an antigen within five 
posure to that antigen. Exposi 
time in the future results in a memory 
response to the second challenge occi 



iers of a species, the spe- 
ity, does not come into 
: challenge to the organism, 
the challenge with a high de- 
the remarkable property of 
i adaptive immune response 
ir six days after the initial ex- 



the 



isponse: the 



e quickly thai 



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the first, is stronger, and is often more effective in neutraliz- 
ing and clearing the pathogen. The major agents of adaptive 
immunity are lymphocytes and the antibodies and other 
molecules they produce. 

Because adaptive immune responses require some time to 
marshal, innate immunity provides the first line of defense 
during the critical period just after the host's exposure to a 
pathogen. In general, most of the microorganisms encoun- 
tered by a healthy individual are readily cleared within a few 
days by defense mechanisms of the inn 
before they activate the adaptive immune syster 



Innate Immunity 



Innate immunity can be seen to comprise four types of de- 
fensive barriers: anatomic, physiologic, phagocytic, and in- 
flammatory (Table 1-2). 

The Skin and the Mucosal Surfaces Provide 
Protective Barriers Against Infection 

Physical and anatomic barriers that tend to prevent the entry 
of pathogens are an organism's first line of defense against in- 
fection. The skin and the surface of mucous membranes are 
included in this category because they are effective barriers to 
the entry of most microorganisms. The skin consists of two 



distinct layers: a thinner outer layer — the epidermis — and a 
thicker layer — the dermis. The epidermis contains several 
layers of tightly packed epithelial cells. The outer epidermal 
layer consists of dead cells and is filled with a waterproofing 
protein called keratin. The dermis, which is composed of 
connective tissue, contains blood vessels, hair follicles, seba- 
ceous glands, and sweat glands. The sebaceous glands are as- 
sociated with the hair follicles and produce an oily secretion 
called sebum. Sebum consists of lactic acid and fatty acids, 
which maintain the pH of the skin between 3 and 5; this pH 
inhibits the growth of most microorganisms. A few bacteria 
that metabolize sebum live as commensals on the skin and 
sometimes cause a severe form of acne. One acne drug, 
isotretinoin (Accutane), is a vitamin A derivative that pre- 
vents the formation of sebum. 

Breaks in the skin resulting from scratches, wounds, or 
abrasion are obvious routes of infection. The skin may also 
be penetrated by biting insects (e.g., mosquitoes, mites, ticks, 
fleas, and sandflies); if these harbor pathogenic organisms, 
they can introduce the pathogen into the body as they feed. 
The protozoan that causes malaria, for example, is deposited 
in humans by mosquitoes when they take a blood meal. Sim- 
ilarly, bubonic plague is spread by the bite of fleas, and Lyme 
disease is spread by the bite of ticks. 

The conjunctivae and the alimentary, respiratory, and 
urogenital tracts are lined by mucous membranes, not by the 
dry, protective skin that covers the exterior of the body. These 



| TABLE 1-2 \ 
Type 


Mechanism 






Anatomic barriers 








Skin 


Mechanical barrier retards entry of microbes. 
Acidic environment (pH 3-5) retards growth of rr 


icrobes. 




Mucous membranes 


Normal flora compete with microbes for attachm 
Mucus entraps foreign microorganisms. 
Cilia propel microorganisms out of body. 


ent sites and nutrients. 




Physiologic barriers 








Temperature 


Normal body temperature inhibits growth of som 
Fever response inhibits growth of some pathogen 


e pathogens. 




LowpH 


Acidity of stomach contents kills most ingested rr 


icroorganisms. 




Chemical mediators 


Lysozyme cleaves bacterial cell wall. 
Interferon induces antiviral state in uninfected ce 
Complement lyses microorganisms or facilitates 
Toll-like receptors recognize microbial molecules 
Collectins disrupt cell wall of pathogen. 


phagocytosis, 
signal cell to secrete immu 


nostimulatory cytokines. 


Phagocytic fendocytic barriers 


Various cells internalize (endocytose) and break c 
Specialized cells (blood monocytes, neutrophils, 
(phagocytose), kill, and digest whole microorga 


own foreign macromolecul 
issue macrophages) intern 


:l 


Inflammatory barriers 


Tissue damage and infection induce leakage of va 
antibacterial activity, and influx of phagocytic ce 


s into the affected area. 


m proteins with 



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membranes consist of an outer epithelial layer and an under- 
lying layer of connective tissue. Although many pathogens 
enter the body by binding to and penetrating mucous mem- 
branes, a number of nonspecific defense mechanisms tend to 
prevent this entry. For example, saliva, t 
cretions act to wash away potential invaders and also ci 
antibacterial or antiviral substances. The viscous fluid called 
mucus, which is secreted by epithelial cells of mucous mem- 
branes, entraps foreign microorganisms. In the lower respi- 
ratory tract, the mucous membrane is covered by cilia, 
hairlike protrusions of the epithelial-cell membranes. The 
synchronous movement of cilia propels mucus-entrapped 
microorganisms from these tracts. In addition, nonpatho- 
genic organisms tend to colonize the epithelial cells of mu- 
cosal surfaces. These normal flora generally outcompete 
pathogens for attachment sites on the epithelial cell surface 
and for necessary nutrients. 

Some organisms have evolved ways of escaping these de- 
fense mechanisms and thus are able to invade the body 
s membranes. For example, influenza virus 
a surface n 



e susceptible to bacterial 



, whereas others 



lecule that enables 
of the respi- 
from being swept out by the 



(the agent that causes flu) ha; 
it to attach firmly to cells in 
ratory tract, preventing the 
ciliated epithelial cells. Similarly, the ( 
gonorrhea has surface projections that allow it to bind to ep- 
ithelial cells in the mucous membrane of the urogenital tract. 
Adherence of bacteria to mucous membranes is due to inter- 
actions between hairlike protrusions on a bacterium, called 
fimbriae or pili, and certain glycoproteins or glycolipids that 
are expressed only by epithelial cells of the 
brane of particular tissues (Figure 1-2). For this n 




1 Electron micrograph of rod-shaped Escherichia coli 
bacteria adhering to surface of epithelial cells of the urinary tract. 
[From N. Sharon and H. Lis, 1993, Sci. Am. 268(1):85; photograph 
courtesy ofK. Fujita.} 



Physiologic Barriers to Infection Include 
General Conditions and Specific Molecules 

The physiologic barriers that contribute to innate immu- 
nity include temperature, pH, and various soluble and cell- 
associated molecules. Many species are not susceptible to cer- 
tain diseases simply because their normal body temperature 
inhibits growth of the pathogens. Chickens, for example, 
have innate immunity to anthrax because their high body 
temperature inhibits the growth of the bacteria. Gastric acid- 
ity is an innate physiologic barrier to infection because very 
few ingested microorganisms can survive the low pH of the 
stomach contents. One reason newborns are susceptible to 
some diseases that do not afflict adults is that their stomach 
contents are less acid than those of adults. 

A variety of soluble factors contribute to innate immu- 
nity, among them the soluble proteins lysozyme, interferon, 
and complement. Lysozyme, a hydrolytic enzyme found in 
mucous secretions and in tears, is able to cleave the peptido- 
glycan layer of the bacterial cell wall. Interferon comprises a 
group of proteins produced by virus-infected cells. Among 
the many functions of the interferons is the ability to bind to 
nearby cells and induce a generalized antiviral state. Comple- 
ment, examined in detail in Chapter 13, is a group of serum 
proteins that circulate in an inactive state. A variety of spe- 
cific and nonspecific immunologic mechanisms can convert 
the inactive forms of complement proteins into an active 
state with the ability to damage the membranes of patho- 
genic organisms, either destroying the pathogens or facilitat- 
ing their clearance. Complement may function as an effector 
system that is triggered by binding of antibodies to certain 
cell surfaces, or it may be activated by reactions between 
complement molecules and certain components of microbial 
cell walls. Reactions between complement molecules or frag- 
ments of complement molecules and cellular receptors trig- 
ger activation of cells of the innate or adaptive immune 
systems. Recent studies on collectins indicate that these sur- 
factant proteins may kill certain bacteria directly by disrupt- 
ing their lipid membranes or, alternatively, by aggregating the 
bacteria to enhance their susceptibility to phagocytosis. 

Many of the molecules involved in innate immunity have 
the property of pattern recognition, the ability to recognize a 
given class of molecules. Because there are certain types of mol- 
ecules that are unique to microbes and never found in multi- 
cellular organisms, the ability to immediately recognize and 
combat invaders displaying such molecules is a strong feature 
of innate immunity. Molecules with pattern recognition ability 
may be soluble, like lysozyme and the complement compo- 
nents described above, or they may be cell-associated receptors. 
Among the class of receptors designated the toll-like receptors 
(TLRs), TLR2 recognizes the lipopolysaccharide (LPS) found 
on Gram-negative bacteria. It has long been recognized that 



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ic46:3 85_reb: 



ing Escherichia coli (green). The bacteria are phagocytized as de- 
scribed in part b and breakdown products secreted. The monocyte 
(purple) has been recruited to the vicinity of the encounter by soluble 
factors secreted by the macrophage. The red sphere is an erythrocyte, 
(b) Schematic diagram of the steps in phagocytosis of a bacterium. 
[Part a, Dennis Kunkel Microscopy, Inc. /Dennis Kunkel.] 



systemic exposure of mammals to relatively small quantities of 
purified LPS leads to an acute inflammatory response (see be- 
low). The mechanism for this response is via a TLR on 
macrophages that recognizes LPS and elicits a variety of mole- 
cules in the inflammatory response upon exposure. When the 
TLR is exposed to the LPS upon local invasion by a Gram-neg- 
ative bacterium, the contained response results in elimination 
of the bacterial challenge. 




Cells That Ingest and Destroy Pathogens 
Make Up a Phagocytic Barrier to Infection 

Another important innate defense mechanism is the inges- 
tion of extracellular particulate material by phagocytosis. 
Phagocytosis is one type of endocytosis, the general term for 
the uptake by a cell of material from its environment. In 
phagocytosis, a cell's plasma membrane expands around the 
particulate material, which may include whole pathogenic 
microorganisms, to form large vesicles called phagosomes 
(Figure 1-3). Most phagocytosis is conducted by specialized 
cells, such as blood monocytes, neutrophils, and tissue 
macrophages (see Chapter 2). Most cell types are capable of 
other forms of endocytosis, such as receptor-mediated endo- 
cytosis, in which extracellular molecules are internalized after 
binding by specific cellular receptors, and pinocytosis, the 
process by which cells take up fluid from the surrounding 
medium along with any molecules contained in it. 

Inflammation Represents a Complex 
Sequence of Events That Stimulates 
Immune Responses 

Tissue damage caused by a wound or by an invading patho- 
genic microorganism induces a complex sequence of events 
collectively known as the inflammatory response. As de- 
scribed above, a molecular component of a microbe, such as 
LPS, may trigger an inflammatory response v 
with cell surface receptors. The end result of inflai 
may be the marshalling of a specific immune response to the 
invasion or clearance of the invader by components of the 
innate immune system. Many of the classic features of the 
inflammatory response were described as early as 1600 bc, in 
Egyptian papyrus writings. In the first century ad, the 
Roman physician Celsus described the "four cardinal signs 



Bacterium becomes attached 
to membrane evaginati 
called pseudopodi 



• : 

J Bacterium is ingested, 
forming phagosome 



Phagosome fuses with 
lysosome 



captured material 




of inflammation" as rubor (redness), tumor (swelling), 
calor (heat), and dolor (pain). In the second century ad, an- 
other physician, Galen, added a fifth sign: functio laesa (loss 
of function). The cardinal signs of inflammation reflect the 
three major events of an inflammatory response (Figure 1-4): 

1. Vasodilation — an increase in the diameter of blood 
vessels — of nearby capillaries occurs as the vessels that 
carry blood away from the affected area constrict, 
resulting in engorgement of the capillary network. The 
engorged capillaries are responsible for tissue redness 
(erythema) and an increase in tissue temperature. 



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Tissue damage causes release of 
vasoactive and chemotactic factors 
that trigger a local increase in blood 
flow and capillary permeability 






Tics and antibacterial 
exudate destroy bacteria 




Major 
rial infection causes tissue damage with release of various vasoactive 
and chemotactic factors. These factors induce increased blood flow 
to the area, increased capillary permeability, and an influx of white 



n the inflammatory response. A bacte- blood cells, including phagocytes and lymphocytes, from the blood 

nage with release of various vasoactive into the tissues. The serum proteins contained in the exudate have 

bacterial properties, and the phagocytes begin to engulf the bac- 

a, as illustrated in Figure 1-3. 



2. An increase in capillary permeability facilitates an influx 
of fluid and cells from the engorged capillaries into the 
tissue. The fluid that accumulates (exudate) has a much 
higher protein content than fluid normally released from 
the vasculature. Accumulation of exudate contributes to 
tissue swelling (edema). 

3. Influx of phagocytes from the capillaries into the tissues is 
facilitated by the increased permeability of the capil- 
laries. The emigration of phagocytes is a multistep 
process that includes adherence of the cells to the 
endothelial wall of the blood vessels (margination), 
followed by their emigration between the capillary- 
endothelial cells into the tissue (diapedesis or extrava- 
sation), and, finally, their migration through the tissue to 
the site of the invasion (chemotaxis). As phagocytic cells 
accumulate at the site and begin to phagocytose bacteria, 
they release lytic enzymes, which can damage nearby 
healthy cells. The accumulation of dead cells, digested 
material, and fluid forms a substance called pus. 

The events in the inflammatory response are initiated by a 
complex series of events involving a variety of chemical me- 
diators whose interactions are only partly understood. Some 
of these mediators are derived from invading microorgan- 



isms, some are released from damaged cells in response to tis- 
sue injury, some are generated by several plasma enzyme sys- 
tems, and some are products of various white blood cells 
participating in the inflammatory response. 

Among the chemical mediators released in response to tis- 
sue damage are various serum proteins called acute-phase 
proteins. The concentrations of these proteins increase dra- 
matically in tissue-damaging infections. C-reactive protein is 
a major acute-phase protein produced by the liver in re- 
sponse to tissue damage. Its name derives from its pattern- 
recognition activity: C-reactive protein binds to the 
C-polysaccharide cell-wall component found on a variety of 
bacteria and fungi. This binding activates the complement 
system, resulting in increased clearance of the pathogen ei- 
ther by complement-mediated lysis or by a complement- 
mediated increase in phagocytosis. 

One of the principal mediators of the inflammatory re- 
sponse is histamine, a chemical released by a variety of cells 
in response to tissue injury. Histamine binds to receptors on 
nearby capillaries and venules, causing vasodilation and in- 
creased permeability. Another important group of inflam- 
matory mediators, small peptides called kinins, are normally 
present in blood plasma in an inactive form. Tissue injury ac- 
tivates these peptides, which then cause vasodilation and in- 



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al . / Immunology 5e 



creased permeability of capillaries. A particular kinin, called 
bradykinin, also stimulates pain receptors in the skin. This 
effect probably serves a protective role, because pain nor- 
mally causes an individual to protect the injured area. 

Vasodilation and the increase in capillary permeability in 
an injured tissue also enable enzymes of the blood-clotting 
system to enter the tissue. These enzymes activate an enzyme 
cascade that results in the deposition of insoluble strands of 
fibrin, which is the main component of a blood clot. The fib- 
rin strands wall off the injured area from the rest of the body 
and serve to prevent the spread of infection. 

Once the inflammatory response has subsided and most 
of the debris has been cleared away by phagocytic cells, tissue 
repair and regeneration of new tissue begins. Capillaries 
grow into the fibrin of a blood clot. New connective tissue 
cells, called fibroblasts, replace the fibrin as the clot dissolves. 
As fibroblasts and capillaries accumulate, scar tissue forms. 
The inflammatory response is described in more detail in 
Chapter 15. 



Adaptive Immunity 



Adaptive immunity is capable of recognizing and selectively 
eliminating specific foreign microorganisms and molecules 
(i.e., foreign antigens). Unlike innate immune responses, 
adaptive immune responses are not the same in all members 
of a species but are reactions to specific antigenic challenges. 
Adaptive immunity displays four characteristic attributes: 

■ Antigenic specificity 

■ Diversity 

■ Immunologic memory 

■ Self/nonself recognition 

The antigenic specificity of the 

distinguish subtle differences ; 
inguish between 



only a singli 
generating trem< 
allowing it to rec 
eign antigens. O 
responded to ar 
that is, a second 
heightened state of 
tribute, the i 
many infectious agent; 






acid. The 
idous diversity 












system permits it to 

Lg antigens. Antibodies 

molecules that differ in 

capable of 

recognition molecules, 

of unique structures on for- 

has recognized and 

t exhibits immunologic memory; 

with the same antigen induces a 

reactivity. Because of this at- 

n confer life-long immunity to 

initial encounter. Finally, the 



n normally responds only to foreign antigen 
indicating that it is capable of self/nonself recognition. The 
ability of the immune system to distinguish self from nonself 
and respond only to nonself molecules is essential, for, as de- 
scribed below, the outcome of an inappropriate response to 
self molecules can be fatal. 

Adaptive immunity is not independent of innate immu- 
nity. The phagocytic cells crucial to nonspecific 



sponses are intimately involved in activating the specific im- 
mune response. Conversely, various soluble factors produced 
by a specific immune response have been shown to augment 
the activity of these phagocytic cells. As an inflammatory re- 
sponse develops, for example, soluble mediators are pro- 
duced that attract cells of the immune system. The immune 
response will, in turn, serve to regulate the intensity of the in- 
flammatory response. Through the carefully regulated inter- 
play of adaptive and innate immunity, the two systems work 
together to eliminate a foreign invader. 

The Adaptive Immune System Requires 
Cooperation Between Lymphocytes and 
Antigen-Presenting Cells 

An effective immune response involves two major groups of 
cells: T lymphocytes and antigen-presenting cells. Lympho- 
cytes are one of many types of white blood cells produced in 
the bone marrow by the process of hematopoiesis (see Chap- 
ter 2). Lymphocytes leave the bone marrow, circulate in the 
blood and lymphatic systems, and reside in various lym- 
phoid organs. Because they produce and display antigen- 
binding cell-surface receptors, lymphocytes mediate the 
defining immunologic attributes of specificity, diversity, 
memory, and self/nonself recognition. The two major popu- 
lations of lymphocytes — B lymphocytes (B cells) and T lym- 
phocytes (T cells) — are described briefly here and in greater 
detail in later chapters. 

B LYMPHOCYTES 

B lymphocytes mature within the bone marrow; when they 
leave it, each expresses a unique antigen-binding receptor on 
its membrane (Figure l-5a). This antigen-binding or B-cell 
receptor is a membrane-bound antibody molecule. Anti- 
bodies are glycoproteins that consist of two identical heavy 
polypeptide chains and two identical light polypeptide 
chains. Each heavy chain is joined with a light chain by disul- 
fide bonds, and additional disulfide bonds hold the two pairs 
together. The amino-terminal ends of the pairs of heavy and 
light chains form a cleft within which antigen binds. When a 
naive B cell (one that has not previously encountered anti- 
gen) first encounters the antigen that matches its membrane- 
bound antibody, the binding of the antigen to the antibody 
causes the cell to divide rapidly; its progeny differentiate into 
memory B cells and effector B cells called plasma cells. 
Memory B cells have a longer life span than naive cells, and 
they express the same membrane-bound antibody as their 
parent B cell. Plasma cells produce the antibody in a form 
that can be secreted and have little or no membrane-bound 
antibody. Although plasma cells live for only a few days, they 
secrete enormous amounts of antibody during this time. 
It has been estimated that a single plasma cell can secrete 
more than 2000 molecules of antibody per second. Secreted 
antibodies are the major effector molecules of humoral 
immunity. 



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(b) T H cell 

1CK 




B cells have about 10 5 molecules of membrane-bound antibody per 
cell. All the antibody molecules on a given B cell have the same anti- 
genic specificity and can interact directly with antigen, (b) T cells 
bearing CD4 (CD4 + cells) recognize only antigen bound to class II 
MHC molecules, (c) T cells bearing CD8 (CD8 + cells) recognize only 



antigen associated with class I MHC molecules. In general, CD4 + 
cells act as helper cells and CD8 + cells act as cytotoxic cells. Both 
types of T cells express about 1 5 identical molecules of the antigen- 
binding T-cell receptor (TCR) per cell, all with the same antigenic 
specificity. 



T LYMPHOCYTES 

T lymphocytes also arise in the bone marrow. Unlike B cells, 
which mature within the bone marrow, T cells migrate to the 
thymus gland to mature. During its maturation within the 
thymus, the T cell comes to express a unique antigen-binding 
molecule, called the T-cell receptor, on its membrane. Unlike 
membrane-bound antibodies on B cells, which can recognize 
antigen alone, T-cell receptors can recognize only antigen 
that is bound to cell-membrane proteins called major histo- 
compatibility complex (MHC) molecules. MHC molecules 
that function in this recognition event, which is termed "anti- 
gen presentation," are polymorphic (genetically diverse) gly- 
coproteins found on cell membranes (see Chapter 7). There 
are two major types of MHC molecules: Class I MHC mole- 
cules, which are expressed by nearly all nucleated cells of ver- 
tebrate species, consist of a heavy chain linked to a small 
invariant protein called (^-microglobulin. Class II MHC 
molecules, which consist of an alpha and a beta glycoprotein 
chain, are expressed only by antigen-presenting cells. When a 
naive T cell encounters antigen combined with a MHC mol- 
ecule on a cell, the T cell proliferates and differentiates into 
memory T cells and various effector T cells. 

There are two well-defined subpopulations of T cells: T 
helper (T H ) and T cytotoxic (T c ) cells. Although a third type 
of T cell, called a T suppressor (T s ) cell, has been postulated, 
recent evidence suggests that it may not be distinct from T H 
and T c subpopulations. T helper and T cytotoxic cells can be 
distinguished from one another by the presence of either 
CD4 or CD8 membrane glycoproteins on their surfaces (Fig- 
ure l-5b,c). T cells displaying CD4 generally function as T H 
cells, whereas those displaying CD8 generally function as T c 
cells (see Chapter 2). 

After a T H cell recognizes and interacts with an anti- 
gen-MHC class II molecule complex, the cell is activated — it 
becomes an effector cell that secretes various growth factors 
known collectively as cytokines. The secreted cytokines play 



an important role in activating B cells, T c cells, macrophages, 
and various other cells that participate in the immune re- 
sponse. Differences in the pattern of cytokines produced by 
activated T H cells result in different types of immune 
response. 

Under the influence of T H -derived cytokines, a T c cell 
that recognizes an antigen-MHC class I molecule complex 
proliferates and differentiates into an effector cell called a cy- 
totoxic T lymphocyte (CTL). In contrast to the T c cell, the 
CTL generally does not secrete many cytokines and instead 
exhibits cell-killing or cytotoxic activity. The CTL has a vital 
function in monitoring the cells of the body and eliminating 
any that display antigen, such as virus-infected cells, tumor 
cells, and cells of a foreign tissue graft. Cells that display for- 
eign antigen complexed with a class I MHC molecule are 
called altered self-cells; these are targets of CTLs. 

ANTIGEN-PRESENTING CELLS 

Activation of both the humoral and cell-mediated branches 
of the immune system requires cytokines produced by T H 
cells. It is essential that activation of T H cells themselves be 
carefully regulated, because an inappropriate T-cell response 
to self-components can have fatal autoimmune conse- 
quences. To ensure carefully regulated activation of T H cells, 
they can recognize only antigen that is displayed together 
with class MHC II molecules on the surface of antigen-pre- 
senting cells (APCs). These specialized cells, which include 
macrophages, B lymphocytes, and dendritic cells, are distin- 
guished by two properties: (1) they express class II MHC 
molecules on their membranes, and (2) they are able to 
deliver a co-stimulatory signal that is necessary for T H -cell 
activation. 

Antigen-presenting cells first internalize antigen, either by 
phagocytosis or by endocytosis, and then display a part of 
that antigen on their membrane bound to a class II MHC 
molecule. The T H cell recognizes and interacts with the 



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' Immunology 5e: 




* Electron micrograph of an antigen-presenting r 
phage (right) associating with a T lymphocyte. [From A. S. Rosenthal 
et at, 1982, in Phagocytosis — Past and Future, Academic Press, p. 



antigen-class II MHC molecule complex on the membrane 
of the antigen-presenting cell (Figure 1-6). An additional co- 
stimulatory signal is then produced by the antigen-present- 
ing cell, leading to activation of the T H cell. 

Humoral Immunity But Not Cellular 
Immunity Is Transferred 
with Antibody 

As mentioned earlier, immune responses can be divided into 
humoral and cell-mediated responses. Humoral immunity 
refers to immunity that can be conferred upon a nonimmune 
individual by administration of serum antibodies from an 
immune individual. In contrast, cell-mediated immunity can 
be transferred only by administration of T cells from an im- 
mune individual. 

The humoral branch of the immune system is at work in 
the interaction of B cells with antigen and their subsequent 
proliferation and differentiation into antibody-secreting 
plasma cells (Figure 1-7). Antibody functions as the effector 
of the humoral response by binding to antigen and neutraliz- 
ing it or facilitating its elimination. When an antigen is 
coated with antibody, it can be eliminated in several ways. 
For example, antibody can cross-link several antigens, form- 
ing clusters that are more readily ingested by phagocytic cells. 
Binding of antibody to antigen on a microorganism can also 
activate the complement system, resulting in lysis of the for- 
eign organism. Antibody can also neutralize toxins or viral 
particles by coating them, which prevents them from binding 
to host cells. 

Effector T cells generated in response to antigen are re- 
sponsible for cell-mediated immunity (see Figure 1-7). Both 



activated T H cells and cytotoxic T lymphocytes (CTLs) serve 
as effector cells in cell-mediated immune reactions. Cy- 
tokines secreted by T H cells can activate various phagocytic 
cells, enabling them to phagocytose and kill microorganisms 
more effectively. This type of cell-mediated immune re- 
sponse is especially important in ridding the host of bacteria 
and protozoa contained by infected host cells. CTLs partici- 
pate in cell-mediated immune reactions by killing altered 
self-cells; they play an important role in the killing of virus- 
infected cells and tumor cells. 

Antigen Is Recognized Differently by 
B and T Lymphocytes 

Antigens, which are generally very large and complex, are not 
recognized in their entirety by lymphocytes. Instead, both B 
and T lymphocytes recognize discrete sites on the antigen 
called antigenic determinants, or epitopes. Epitopes are the 
immunologically active regions on a complex antigen, the re- 
gions that actually bind to B-cell or T-cell receptors. 

Although B cells can recognize an epitope alone, T cells 
can recognize an epitope only when it is associated with an 
MHC molecule on the surface of a self-cell (either an anti- 
gen-presenting cell or an altered self-cell). Each branch of the 
immune system is therefore uniquely suited to recognize 
antigen in a different milieu. The humoral branch (B cells) 
recognizes an enormous variety of epitopes: those displayed 
on the surfaces of bacteria or viral particles, as well as those 
displayed on soluble proteins, glycoproteins, polysaccha- 
rides, or lipopolysaccharides that have been released from in- 
vading pathogens. The cell-mediated branch (T cells) 
recognizes protein epitopes displayed together with MHC 
molecules on self-cells, including altered self-cells such as 
virus-infected self-cells and cancerous cells. 

Thus, four related but distinct cell-membrane molecules 
are responsible for antigen recognition by the immune 
system: 

■ Membrane-bound antibodies on B cells 

■ T-cell receptors 

« Class I MHC molecules 

■ Class II MHC molecules 

Each of these molecules plays a unique role in antigen recog- 
nition, ensuring that the immune system can recognize and 
respond to the different types of antigen that it 



B and T Lymphocytes Utilize Similar 
Mechanisms To Generate Diversity 
in Antigen Receptors 

The antigenic specificity of each B cell is determined by the 
membrane-bound antigen-binding receptor (i.e., antibody) 
expressed by the cell. As a B cell matures in the bone marrow, 
its specificity is created by random rearrangements of a series 



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' Immunology 5e: 



VISUALIZING CONCEPTS 



Foreign 
proteins 


Antigens 
Viruses Bacteria Parasites 


l^gT 




branches of the immune system. In the humoral response, B cells 
interact with antigen and then differentiate into antibody-secret- 
ing plasma cells. The secreted antibody binds to the antigen and 
facilitates its clearance from the body. In the cell-mediated re- 



sponse, various subpopulations of T cells recognize antigen pre- 
sented on self-cells. T H cells respond to antigen by producing cy- 
tokines. T c cells respond to antigen by developing into cytotoxic T 
lymphocytes (CTLs), which mediate killing of altered self-cells 
(e.g., virus-infected cells). 



A 



i01_013 9/5/02 



of gene segments that encode the antibody molecule (see 
Chapter 5). As a result of this process, each mature B cell pos- 
sesses a single functional gene encoding the antibody heavy 
chain and a single functional gene encoding the antibody 
light chain; the cell therefore synthesizes and displays anti- 
body with one specificity on its membrane. All antibody 
molecules on a given B lymphocyte have identical specificity, 
giving each B lymphocyte, and the clone of daughter cells to 
which it gives rise, a distinct specificity for a single epitope on 
an antigen. The mature B lymphocyte is therefore said to be 
antigenically committed. 

The random gene rearrangements during B-cell matura- 



n the b 



-nber of 



different antigenic specificities. The resulting B-cell popula- 
tion, which consists of individual B cells each expressing a 
unique antibody, is estimated to exhibit collectively more 
than 10 10 different antigenic specificities. The enormous di- 
versity in the mature B-cell population is later reduced by a 
selection process in the bone marrow that eliminates any B 
cells with membrane-bound antibody that recognizes self- 
components. The selection process helps to ensure that self- 
reactive antibodies (auto-antibodies) are not produced. 

The attributes of specificity and diversity also characterize 
the antigen-binding T-cell receptor (TCR) on T cells. As in B- 
cell maturation, the process of T-cell maturation includes 
random rearrangements of a series of gene segments that en- 
code the cell's antigen-binding receptor (see Chapter 9). Each 
T lymphocyte cell expresses about 10 5 receptors, and all of 
the receptors on the cell and its clonal progeny have identical 
specificity for antigen. The random rearrangement of the 



TCR genes is capable of generating on the order of 10 
unique antigenic specificities. This enormous potential di- 
versity is later diminished through a selection process in the 
thymus that eliminates any T cell with self-reactive receptors 
and ensures that only T cells with receptors capable of recog- 
nizing antigen associated with MHC molecules will be able 
to mature (see Chapter 10). 



The Major Histocompatibility Molecules 
Bind Antigenic Peptides 

The major histocompatibility complex (MHC) is a large ge- 
netic complex with multiple loci. The MHC loci encode two 
major classes of membrane-bound glycoproteins: class I and 
class II MHC molecules. As noted above, T H cells generally 
recognize antigen combined with class II molecules, whereas 
T c cells generally recognize antigen combined with class I 
molecules (Figure 1-8). 

MHC molecules function as antigen-recognition mole- 
cules, but they do not possess the fine specificity for antigen 
characteristic of antibodies and T-cell receptors. Rather, each 
MHC molecule can bind to a spectrum of antigenic peptides 
derived from the intracellular degradation of antigen mole- 
cules. In both class I and class II MHC molecules the distal 
regions (farthest from the membrane) of different alleles dis- 
play wide variation in their amino acid sequences. These 
variable regions form a cleft within which the antigenic pep- 
tide sits and is presented to T lymphocytes (see Figure 1-8). 
Different allelic forms of the genes encoding class I and class 




ffl TheroleofMHCm 


olecules in antigen recognition by 


T cells, (a) Class 1 MHC molecules 


are expressed on nearly all nude- 


ated cells. Class II MHC molecule 


s are expressed only on antigen- 


presenting cells. T cells that rec 


ognize only antigenic peptides 


displayed with a class II MHC mole 


;ule generally function as T helper 


(T H ) cells. T cells that recognize c 


nly antigenic peptides displayed 


with a class 1 MHC molecule gene 


rally function as T cytotoxic (T c ) 



. (b) This 



rograph n 



lymphocytes interacting with a single macrophage. The macrophage 
presents processed antigen combined with class II MHC molecules 
to the T cells. [Photograph from W. E. Paul (ed.), 1991, Immunology: 
Recognition and Response, W. H. Freeman and Company, New York; 
micrograph courtesy ofM. H. Nielsen and O. Werdelin.] 



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II molecules confer different structures on the antigen-bind- 
ing cleft with different specificity. Thus the ability to present 
an antigen to T lymphocytes is influenced by the particular 
set of alleles that an individual inherits. 

Complex Antigens Are Degraded (Processed) 
and Displayed (Presented) with MHC 
Molecules on the Cell Surface 

In order for a foreign protein antigen to be recognized by a T 
cell, it must be degraded into small antigenic peptides that 
form complexes with class I or class II MHC molecules. This 
conversion of proteins into MHC-associated peptide frag- 
ments is called antigen processing and presentation. Whether a 
particular antigen will be processed and presented together 
with class I MHC or class II MHC molecules appears to be 
determined by the route that the antigen takes to enter a cell 
(Figure 1-9). 

Exogenous antigen is produced outside of the host cell 
and enters the cell by endocytosis or phagocytosis. Antigen- 
presenting cells (macrophages, dendritic cells, and B cells) 
degrade ingested exogenous antigen into peptide fragments 
within the endocytic processing pathway. Experiments sug- 
gest that class II MHC molecules are expressed within the en- 
docytic processing pathway and that peptides produced by 
degradation of antigen in this pathway bind to the cleft 
within the class II MHC molecules. The MHC molecules 
bearing the peptide are then exported to the cell surface. 



Since expression of class II MHC molecules is limited to anti- 
gen-presenting cells, presentation of exogenous peptide- 
class II MHC complexes is limited to these cells. T cells dis- 
playing CD4 recognize antigen combined with class II MHC 
molecules and thus are said to be class II MHC restricted. 
These cells generally function as T helper cells. 

Endogenous antigen is produced within the host cell it- 
self. Two common examples are viral proteins synthesized 
within virus-infected host cells and unique proteins synthe- 
sized by cancerous cells. Endogenous antigens are degraded 
into peptide fragments that bind to class I MHC molecules 
within the endoplasmic reticulum. The peptide-class I MHC 
complex is then transported to the cell membrane. Since all 
nucleated cells express class I MHC molecules, all cells pro- 
ducing endogenous antigen use this route to process the anti- 
gen. T cells displaying CD8 recognize antigen associated with 
class I MHC molecules and thus are said to be class I MHC re- 
stricted. These cytotoxic T cells attack and kill cells displaying 
the antigen-MHC class I complexes for which their receptors 

Antigen Selection of Lymphocytes 
Causes Clonal Expansion 

A mature immunocompetent animal contains a large num- 
ber of antigen-reactive clones of T and B lymphocytes; the 
antigenic specificity of each of these clones is determined by 
the specificity of the antigen-binding receptor on the mem- 



Peptide-class I MHC complex 

Class I MHC 

iral peptide 

Vesicle 




' Processing and presentation of exoger 



and e 



dogenous antigens, (a) Exogenc 
sis or phagocytosis and ther 
pathway. Here, within an acidic 6 
into small peptides, which then ; 






n [he 



is antigen is ingested by endocyto- 
enters the endocytic processing 
vironment, the antigen is degraded 
; presented with class II MHC mol- 



e of the antigen-presenting cell, (b) Endoge- 



nous antigen, which is produced within the cell itself (e.g., in a virus- 
infected cell), is degraded within the cytoplasm into peptides, which 
move into the endoplasmic reticulum, where they bind to class I 
MHC molecules. The peptide-class I MHC complexes then move 
through the Colgi complex to the cell surface. 



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' Immunology 5e: 



brane of the clone's lymphocytes. As noted above, the speci- 
ficity of each T and B lymphocyte is determined before its 
contact with antigen by random ger 
maturation in the thymus or b> 

The role of antigen becomes critical when it 
and activates mature, antigenically committed T and B lym- 
phocytes, bringing about expansion of the population of 
cells with a given antigenic specificity. In this process of 
clonal selection, an antigen binds to a particular T or B cell 
and stimulates it to divide repeatedly into a clone of cells with 
the same antigenic specificity as the original parent cell (Fig- 
ure 1-10). 

Clonal selection provides a framework for understanding 
the specificity and self/nonself recognition that is character- 



istic of adaptive immunity. Specificity is shown because only 
lymphocytes whose receptors are specific for a given epitope 
on an antigen will be clonally expanded and thus mobilized 
for an immune response. Self/nonself discrimination is ac- 
complished by the elimination, during development, of lym- 
phocytes bearing self-reactive receptors or by the functional 
suppression of these cells in adults. 

Immunologic memory also is a consequence of clonal se- 
lection. During clonal selection, the number of lymphocytes 
specific for a given antigen is greatly amplified. Moreover, 
many of these lymphocytes, referred to as memory cells, ap- 
pear to have a longer life span than the naive lymphocytes 
from which they arise. The initial encounter of a naive im- 
ipetent lymphocyte with an antigen induces a 



Peripheral lyr: 




Maturation into mature 
antigenetically committed B cells 



Antigen-dependent proliferation and 
differentiation into plasma and memory cells 



and clonal selection of B lymphocytes, 
i, which occurs in the absence of antigen, produces anti- 
genically committed B cells, each of which expresses antibody with a 
single antigenic specificity (indicated by 1, 2, 3, and 4). Clonal selec- 
tion occurs when an antigen binds to a B cell whose membrane- 
bound antibody molecules are specific for epitopes on that antigen. 
Clonal expansion of an antigen-activated B cell (number 2 in this ex- 



ample) leads to a clone of memory B cells and effector B cells, called 
plasma cells; all cells in the expanded clone are specific for the orig- 

vating antigen. Similar processes take place in the T-lymphocyte 
population, resulting in clones of memory T cells and effector T cells; 
the latter include activated T H cells, which secrete cytokines, and cy- 
totoxic! lymphocytes (CTLs). 



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primary response; a later contact of the host with antigen 
will induce a more rapid and heightened secondary re- 
sponse. The amplified population of memory cells accounts 
for the rapidity and intensity that distinguishes a secondary 
response from the primary response. 

In the humoral branch of the immune system, antigen in- 
duces the clonal proliferation of B lymphocytes into anti- 




| Differences in the primary and secondary response 
to injected antigen (humoral response) and to a skin graft (cell-me- 
diated response) reflect the phenomenon of immunologic memory, 
(a) When an animal is injected with an antigen, it produces a primary 
serum antibody response of low magnitude and short duration, 
peaking at about 10-17 days. A second immunization with the same 
antigen results in a secondary response that is greater in magnitude, 
peaks in less time (2-7 days), and lasts longer (months to years) 
than the primary response. Compare the secondary response to anti- 
gen A with the primary response to antigen B administered to the 
same mice, (b) Results from a hypothetical experiment in which skin 
grafts from strain C mice are transplanted to 20 mice of strain A; the 
grafts are rejected in about 10-i4 days. The 20 mice are rested for 2 
months and then 10 are given strain C grafts and the other 10 are 
given skin from strain B. Mice previously exposed to strain C skin re- 
ject C grafts much more vigorously and rapidly than the grafts from 
strain B. Note that the rejection of the B graft follows a time course 
similar to that of the first strain C graft. 



body-secreting plasma cells and memory B cells. As seen in 
Figure 1-1 la, the primary response has a lag of approxi- 
mately 5-7 days before antibody levels start to rise. This lag is 
the time required for activation of naive B cells by antigen 
and T H cells and for the subsequent proliferation and differ- 
entiation of the activated B cells into plasma cells. Antibody 
levels peak in the primary response at about day 14 and then 
begin to drop off as the plasma cells begin to die. In the 
secondary response, the lag is much shorter (only 1-2 days), 
antibody levels are much higher, and they are sustained for 
much longer. The secondary response reflects the activity 
of the clonally expanded population of memory B cells. 
These memory cells respond to the antigen more rapidly 
than naive B cells; in addition, because there are many 
more memory cells than there were naive B cells for the 
primary response, more plasma cells are generated in the 
secondary response, and antibody levels are consequently 
100- to 1000-fold higher. 

In the cell-mediated branch of the immune system, the 
recognition of an antigen-MHC complex by a specific ma- 
ture T lymphocyte induces clonal proliferation into various 
T cells with effector functions (T H cells and CTLs) and into 
memory T cells. The cell-mediated response to a skin graft is 
illustrated in Figure 1-1 lb by a hypothetical transplantation 
experiment. When skin from strain C mice is grafted onto 
strain A mice, a primary response develops and all the grafts 
are rejected in about 10-14 days. If these same mice are again 
grafted with strain C skin, it is rejected much more vigor- 
ously and rapidly than the first grafts. However, if animals 
previously engrafted with strain C skin are next given skin 
from an unrelated strain, strain B, the response to strain B is 
typical of the primary response and is rejected in 10-14 days. 
That is, graft rejection is a specific immune response. The 
same mice that showed a secondary response to graft C will 
show a primary response to graft B. The increased speed of 
rejection of graft C reflects the presence of a clonally ex- 
panded population of memory T H and T c cells specific for 
the antigens of the foreign graft. This expanded memory 
population generates more effector cells, resulting in faster 
graft rejection. 

The Innate and Adaptive Immune Systems 
Collaborate, Increasing the Efficiency of 
Immune Responsiveness 

It is important to appreciate that adaptive and innate immu- 
nity do not operate independently — they function as a highly 
interactive and cooperative system, producing a combined 
response more effective than either branch could produce by 
itself. Certain immune components play important roles in 
both types of immunity. 

An example of cooperation is seen in the encounter 
between macrophages and microbes. Interactions between 
receptors on macrophages and microbial components gen- 
erate soluble proteins that stimulate and direct adaptive im- 
mune responses, facilitating the participation of the adap- 



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TABLE 1-3 



Response to Identical to Much more rapid thar 

repeat primary primary response 

infection response 



tive immune system in the elimination of the pathogen. 
Stimulated macrophages also secrete cytokines that can 
direct adaptive immune responses against particular intra- 
cellular pathogens. 

Just as important, the adaptive immune system produces 
signals and components that stimulate and increase the ef- 
fectiveness of innate responses. Some T cells, when they en- 
counter appropriately presented antigen, synthesize and 
secrete cytokines that increase the ability of macrophages to 
kill the microbes they have ingested. Also, antibodies pro- 
duced against an invader bind to the pathogen, marking it as 
a target for attack by complement and serving as a potent ac- 
tivator of the attack. 

A major difference between adaptive and innate immu- 
nity is the rapidity of the innate immune response, which uti- 
lizes a pre-existing but limited repertoire of responding 
components. Adaptive immunity compensates for its slower 
onset by its ability to recognize a much wider repertoire of 
foreign substances, and also by its ability to improve during a 
response, whereas innate immunity remains constant. It may 
also be noted that secondary adaptive responses are consid- 
erably faster than primary responses. Principle characteris- 
tics of the innate and adaptive immune systems are 
compared in Table 1-3. With overlapping roles, the two sys- 
tems together form a highly effective barrier to infection. 



Comparative Immunity 

The field of immunology is concerned mostly with how in- 
nate and adaptive mechanisms collaborate to protect verte- 
brates from infection. Although many cellular and molecular 
actors have important roles, antibodies and lymphocytes are 
considered to be the principal players. Yet despite their 
prominence in vertebrate immune systems, it would be a 
mistake to conclude that these extraordinary molecules and 
versatile cells are essential for immunity. In fact, a deter- 
mined search for antibodies, T cells, and B cells in organisms 
of the nonvertebrate phyla has failed to find them. The inte- 
rior spaces of organisms as diverse as fruit flies, cockroaches, 
and plants do not contain unchecked microbial populations, 



however, which implies that some sort of ir 

most, possibly all, multicellular organisms, including those 

with no components of adaptive immunity. 

Insects and plants provide particularly clear and dramatic 
examples of innate immunity that is not based on lympho- 
cytes. The invasion of the interior body cavity of the fruit fly, 
Drosophila melanogaster, by bacteria or molds triggers the 
synthesis of small peptides that have strong antibacterial or 
antifungal activity. The effectiveness of these antimicrobial 
peptides is demonstrated by the fate of mutants that are un- 
able to produce them. For example, a fungal infection over- 
whelms a mutant fruit fly that is unable to trigger the 
synthesis of drosomycin, an antifungal peptide (Figure 
1-12). Further evidence for immunity in the fruit fly is given 
by the recent findings that cell receptors recognizing various 
classes of microbial molecules (the toll-like receptors) were 
first found in Drosophila. 

Plants respond to infection by producing a wide variety 
of antimicrobial proteins and peptides, as well as small 




P 6 ' Severe fungal infection in a fruit fly (Drosophila 
melanogaster) with a disabling mutation in a signal-transduction 
pathway required for the synthesis of the antifungal peptide dro- 
somycin. [From B. Lemaitre et al., 1996, Cell 86:973; courtesy of J. A. 
Hoffman, University of Strasbourg.] 



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nonpeptide organic molecules that have antibiotic activity. 
Among these agents are enzymes that digest microbial cell 
walls, peptides and a protein that damages microbial mem- 
branes, and the small organic molecules phytoalexins. The 
importance of the phytoalexins is shown by the fact that mu- 
tations that alter their biosynthetic pathways result in loss of 
resistance to many plant pathogens. In some cases, the re- 
sponse of plants to pathogens goes beyond this chemical as- 
sault to include an architectural response, in which the plant 
isolates cells in the infected area by strengthening the walls of 
surrounding cells. Table 1-4 compares the capabilities of im- 
mune systems in a wide range of multicellular organisms, 
both animals and plants. 



Immune Dysfunction and 
Its Consequences 

The above overview of innate and adaptive immunity depicts 
a multicomponent interactive system that protects the host 
from infectious diseases and from cancer. This overview 
would not be complete without mentioning that the immune 
system can function improperly. Sometimes the immune sys- 
tem fails to protect the host adequately or misdirects its ac- 
tivities to cause discomfort, debilitating disease, or even 
death. There are several common manifestations of immune 
dysfunction: 

■ Allergy and asthma 

■ Graft rejection and graft- versus-host disease 

■ Autoimmune disease 

■ Immunodeficiency 

Allergy and asthma are results of inappropriate immune re- 
sponses, often to common antigens such as plant pollen, 
food, or animal dander. The possibility that certain sub- 
stances increased sensitivity rather than protection was rec- 
ognized in about 1902 by Charles Richet, who attempted to 
immunize dogs against the toxins of a type of jellyfish, 
Physalia. He and his colleague Paul Portier observed that 
dogs exposed to sublethal doses of the toxin reacted almost 
instantly, and fatally, to subsequent challenge with minute 
amounts of the toxin. Richet concluded that a successful im- 
munization or vaccination results in phylaxis, or protection, 
and that an opposite result may occur — anaphylaxis — in 
which exposure to antigen can result in a potentially lethal 
sensitivity to the antigen if the exposure is repeated. Richet 
received the Nobel Prize in 1913 for his discovery of the ana- 
phylactic response. 

Fortunately, most allergic reactions in humans are not 
rapidly fatal. A specific allergic or anaphylactic response usu- 
ally involves one antibody type, called IgE. Binding of IgE to 
its specific antigen (allergen) releases substances that cause 
irritation and inflammation. When an allergic individual is 
exposed to an allergen, symptoms may include ; 



wheezing, and difficulty in breathing (asthma); dermatitis or 
skin eruptions (hives); and, in more extreme cases, strangu- 
lation due to blockage of airways by inflammation. A signifi- 
cant fraction of our health resources is expended to care for 
those suffering from allergy and asthma. The frequency of 
allergy and asthma in the United States place these com- 
plaints among the most common reasons for a visit to the 
doctor's office or to the hospital emergency room (see Clini- 
cal Focus). 

When the immune system encounters foreign cells or tis- 
sue, it responds strongly to rid the host of the invaders. How- 
ever, in some cases, the transplantation of cells or an organ 
from another individual, although viewed by the immune 
system as a foreign invasion, may be the only possible treat- 
ment for disease. For example, it is estimated that more than 
60,000 persons in the United States alone could benefit from 
a kidney transplant. Because the immune system will attack 
and reject any transplanted organ that it does not recognize 
as self, it is a serious barrier to this potentially life-saving 
treatment. An additional danger in transplantation is that 
any transplanted cells with immune function may view the 
new host as nonself and react against it. This reaction, which 
is termed graft-versus-host disease, can be fatal. The rejec- 
tion reaction and graft-versus-host disease can be suppressed 
by drugs, but this type of treatment suppresses all immune 
function, so that the host is no longer protected by its im- 
mune system and becomes susceptible to infectious diseases. 
Transplantation studies have played a major role in the de- 
velopment of immunology. A Nobel prize was awarded to 
Karl Landsteiner, in 1930, for the discovery of human blood 
groups, a finding that allowed blood transfusions to be car- 
ried out safely. In 1980, G. Snell, J. Dausset, and B. Benacerraf 
were recognized for discovery of the major histocompatibil- 
ity complex, and, in 1991, E. D. Thomas and J. Murray were 
awarded Nobel Prizes for advances in transplantation immu- 
nity. To enable a foreign organ to be accepted without sup- 
pressing immunity to all antigens remains a challenge for 
immunologists today. 

In certain individuals, the immune system malfunctions 
by losing its sense of self and nonself, which permits an im- 
mune attack upon the host. This condition, autoimmunity, 
can cause a number of chronic debilitating diseases. The 
symptoms of autoimmunity differ depending on which 
tissues and organs are under attack. For example, multiple 
sclerosis is due to an autoimmune attack on the brain and 
central nervous system, Crohn's disease is an attack on the 
tissues in the gut, and rheumatoid arthritis is an attack on 
joints of the arms and legs. The genetic and environmental 
factors that trigger and sustain autoimmune disease are very 
active areas of immunologic research, as is the search for im- 
proved treatments. 

If any of the many components of innate or specific im- 
munity is defective because of genetic abnormality, or if any 
immune function is lost because of damage by chemical, 
physical, or biological agents, the host suffers from immu- 
nodeficiency. The severity of the immunodeficiency disease 



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immunity 



BB9SBB 

Invasion- 
induced 
protective 
Adaptive enzymes 
immunity and enzyme 



Taxonomic group (nonspecific) (specific) cascades Phagocyt 

Higher plants + + 

Invertebrate animals 

Porifera + ? + 

(sponges) 



Antimicrobial recognition Craft T and B 

peptides receptors rejection cells Antibodies 



Arthropods 
(insects, 
crustaceans) 

Vertebrate animals 

Elasmobranchs 
(cartilaginous 
fish; e.g., 
sharks, rays) 

Teleost fish and 
bony fish (e.g., 
salmon, tuna) 

Amphibians 

Reptiles 

Birds 

Mammals 



SOURCES: L Dl 
W. E. Paul (ed.), 
1998, Curr. Opin 






CLINICAL FOCUS 



Allergy and Asthma as Serious 
Public Health Problems 



Although the im 

mune system serves to protect the host 
from infection and cancer, inappropriate 
responses of this system can lead to 
disease. Common among the results of 
immune dysfunction are allergies and 
asthma, both serious public health prob- 



lems. Details of the mechanisms that un- 
derlie allergic and asthmatic responses 
to environmental antigens (or allergens) 
will be considered in Chapter 16. Simply 
stated, allergic reactions are responses 
to antigenic stimuli that result in immu- 
nity based mainly on the IgE class of im- 
munoglobulin. Exposure to the antigen 



(or allergen) triggers an IgE-mediated re- 
lease of molecules that cause symptoms 
ranging from sneezing and dermatitis to 
inflammation of the lungs in an asth- 
matic attack. The sequence of events in 
an allergic response is depicted in the ac- 
companying figure. 

The discomfort from common aller- 
gies such as plant pollen allergy (often 
called ragweed allergy) consists of a 
week or two of sneezing and runny nose, 
which may seem trivial compared with 
health problems such as cancer, cardiac 
arrest, or life-threatening infections. A 
more serious allergic reaction is asthma, 
(continued) 



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i AM Page 2 n 



CLINICAL FOCUS (continued) 



Allergy and Asthma as Serious 
Public Health Problems 



:ase of the lungs in which 
mediated by environmen- 
tal antigens or infections, causes severe 
difficulty in breathing. Approximately 15 
million persons in the United States suf- 
fer from asthma, and it causes about 
5000 deaths per year. In the past twenty 
years, the prevalence of asthma in the 
Western World has doubled.* 

Data on the frequency of care sought 
for the most common medical com- 
plaints in the United States show that 
asthma and allergy together resulted in 
more than 28 million visits to the doctor 
in 1995. The importance of allergy as a 
public health problem is underscored by 
the fact that the annu; 
visits for hypertensio 
examinations, or nor 
each fewer than the r 
allergic conditions. 



iber of doctor 
routine medical 
il pregnancy, are 
nber of visits for 
fact, the most 
common reason for a visit to a hospital 
emergency room is an asthma attack, ac- 
counting for one third of all visits. In ad- 
dition to those treated in the ER, there 
were about 160,000 hospitalizations for 
asthma in the past year, with an average 
stay of 3 to 4 days. 

Although all ages and races are af- 
fected, deaths from asthma are 3.5 times 
more common among African-American 
children. The reasons for the increases in 
number of asthma cases and for the 
higher death rate in African-American chil- 
dren remain unknown, although some 
clues may have been uncovered by recent 



studies of genetic factors in allergic dis- 
ease (see Clinical Focus in Chapter 16). 

An increasingly serious health prob- 
lem is food allergy, especially to peanuts 
and tree nuts (almonds, cashews, and 
walnuts). 1 " Approximately 3 million 
Americans are allergic to these foods 
and they are the leading causes of fatal 
and near-fatal food allergic (anaphylac- 
tic) reactions. While avoidance of these 
foods can prevent harmful conse- 
quences, the ubiquitous use of peanut 
protein and other nut products in a vari- 
ety of foods makes this very difficult for 
the allergic individual. At least 50% of se- 
rious reactions are caused by accidental 
exposures to peanuts, tree nuts, or their 
products. This has led to controversial 
movements to ban peanuts from 
schools and airplanes. 

Anaphylaxis generally occurs within 
an hour of ingesting the food allergen 
and the most effective treatment is injec- 
tion of the drug epinephrine. Those 
prone to anaphylactic attacks often carry 
injectable epinephrine to be used in case 
of exposure. 

In addition to the suffering and anxi- 
ety caused by inappropriate immune re- 
sponses or allergies to environmental 
antigens, there is a staggering cost in 
terms of lost work time for those affected 
and for caregivers. These costs well justify 
the extensive efforts by basic and clinical 
immunologists and allergists to relieve 
the suffering caused by these disorders. 



First contact 

■with an allergen (ragweed) 




Productio 
=C large ai 

of ragweed IgE 





st (London) 48:201. 



IgE-primed mast 
cell releases 
molecules that 
cause wheezing, 
sneezing, runny no 
watery eyes, and 
other symptoms 



Sequence of events leading to an allergic 
response. When the antibody produced 
upon contact with an allergen is IgE, this 
class of antibody reacts via its constant 
region with a mast cell. Subsequent reac- 
tion of the antibody binding site with the 
allergen triggers the mast cell to which 
the IgE is bound to secrete molecules 
that cause the allergic symptoms. 



depends on the number of affected components. A c( 
type of immunodeficiency in North America is a selective 
immunodeficiency in which only one type of immunoglob- 
ulin, IgA, is lacking; the symptoms may be minor or even go 
unnoticed. In contrast, a rarer immunodeficiency called 



severe combined immunodeficiency (SCID), which affects 
both B and T cells, if untreated, results in death from infec- 
tion at an early age. Since the 1980s, the most common form 
of immunodeficiency has been acquired immune deficiency 
syndrome, or AIDS, which results from infection with the 



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' Immunology 5e: 



s human immunodeficiency virus, or HIV. In AIDS, 
T helper cells are infected and destroyed by HIV, causing a 
collapse of the immune system. It is estimated that 35 million 
persons worldwide suffer from this disease, which is usually 
fatal within 8 to 10 years after infection. Although certain 
treatments can prolong the life of AIDS patients, there is no 
known cure for this disease. 

l a brief introduction to the immune 

a thumbnail sketch of how this com- 

i protect the host from disease. The 

e structure and function of 

Lolecules that make up this 

rent understanding of how 

md the experiments 



This chapter has beei 
system, and it has given 
plex system functions t< 
following chapl 
the individual cells, organ* 
system. They will describe 
the components of 



that allowed discovery of thes 
applied immunology, such a 



e mechanisms. Specific a 
is immunity to infectious dis- 
n practices are the subject 
matter of later chapters. Finally, to complete the description 
of the immune system in all of its activities, a chapter ad- 
dresses each of the major types of immune dysfur 



SUMMARY 

■ Immunity is the state of protection against foreign organ- 
isms or substances (antigens). Vertebrates have two types 
of immunity, innate and adaptive. 

■ Innate immunity is not specific to any one pathogen but 
rather constitutes a first line of defense, which includes 
anatomic, physiologic, endocytic and phagocytic, and in- 
flammatory barriers. 

■ Innate and adaptive immunity operate in cooperative and 
interdependent ways. The activation of innate immune re- 
sponses produces signals that stimulate and direct subse- 
quent adaptive immune responses. 

■ Adaptive immune responses exhibit four immunologic at- 
tributes: specificity, diversity, memory, and self/nonself 
recognition. 

■ The high degree of specificity in adaptive immunity arises 
from the activities of molecules (antibodies and T-cell 
receptors) that recognize and bind specific antigens. 

■ Antibodies recognize and interact directly with antigen. T- 
cell receptors recognize only antigen that is combined with 
either class I or class II major histocompatibility complex 
(MHC) molecules. 

■ The two major subpopulations of T lymphocytes are the 
CD4 + T helper (T H ) cells and CD8 + T cytotoxic (T c ) cells. 
T H cells secrete cytokines that regulate immune response 
upon recognizing antigen combined with class II MHC. T c 
cells recognize antigen combined with class I MHC and 
give rise to cytotoxic T cells (CTLs), which display cyto- 
toxic ability. 

■ Exogenous (extracellular) antigens are internalized and 
degraded by antigen-presenting cells (macrophages, B 



cells, and dendritic cells); the resulting antigenic peptides 
complexed with class II MHC molecules are then displayed 
on the cell surface, 
i Endogenous (intracellular) antigens (e.g., viral and tumor 
proteins produced in altered self-cells) are degraded in the 
cytoplasm and then displayed with class I MHC molecules 
on the cell surface. 

■ The immune system produces both humoral and cell-me- 
diated responses. The humoral response is best suited for 
elimination of exogenous antigens; the cell-mediated re- 
sponse, for elimination of endogenous antigens. 

i While an adaptive immune system is found only in verte- 
brates, innate immunity has been demonstrated in organ- 
isms as different as insects, earthworms, and higher plants. 

■ Dysfunctions of the immune system include common 
maladies such as allergy or asthma. Loss of immune func- 
tion leaves the host susceptible to infection; 
nity, the immune system attacks host cells o 



References 

Akira, S., K. Takeda, and T. Kaisho. 2001. Toll-like receptors: 
Critical proteins linking innate and acquired immunity. Na- 
ture Immune 

Burnet, F. M. 1959. The Clonal Selection Theory of Acquired Im- 
munity. Cambridge University Press, Cambridge. 

Cohen, S. C, and M. Samter. 1992. Excerpts from Classics in Al- 
lergy. Symposia Foundation, Carlsbad, Ca 

Desour, L. 1922. Pasteur and His Work (translated by A. F. and 
B. H. Wedd). T. Fisher Unwin Ltd., London. 

Fritig, B., T. Heitz, and M. Legrand. 1998. Antimicrobial proteins 
in induced plant defense. Curr. Opin. Immunol. 10:12. 

Kimbrell, D. A., and B. Beutler. 2001. The evolution and 
genetics of innate immunity. Nature Rev. Genet. 2:256. 

Kindt, T. J., and J. D. Capra. 1984. The Antibody Enigma. 
Plenum Press, New York. 

Landsteiner, K. 1947. 77/. Reactions. Har- 

vard University Press, Cambridge, Massachusetts. 

Lawson, P. R., and K. B. Reid. 2000. The roles of surfactant 
proteins A and D in innate immunity. Immunologic Reviews 
173:66. 

Medawar, P. B. 1958. The Immiin Imitation. The 

Harvey Lectures 1956-1957. Academic Press, New York. 

Medzhitov, R., and C. A. Janeway. 2000. Innate immunity. N. 
Eng. J. Med. 343:338. 



the Infectious Diseases. 



Metchnikoff, E. 1905. Immunity i 
MacMillan, New York. 

Otvos, L. 2000. Antibacterial peptides isolated from insects. /. 
Peptide Sci. 6:497. 

Paul, W., ed. 1999. Funt mology, 4th ed. Lippin- 

cott-Raven, Philadelphia. 



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49 AM Page 22 n 



Roitt, I. M., and P. J. Delves, eds. 1998. An Encyclopedia of Im- 
munology, 2nd ed., vols. 1-4. Academic Press, London. 



EFULWEB SITES 



The American Academy of Allergy Asthma and Immunology 
site includes an extensive library of information about allergic 
diseases. 

http://l 2.1 7.1 2.70/aai/default.asp 

The Web site of the American Association of Immunologists 
od deal of information of interest to immunolo- 



http://www.ncbi.nlm.nih.gov/PubMed/ 

PubMed, the National Library of Medicine database of more 
than 9 million publications, is the world's most comprehen- 
sive bibliographic database for biological and biomedical lit- 
erature. It is also a highly user-friendly site. 



Study Questions 



Clinical Focus Question You have a young nephew who has 
developed a severe allergy to tree nuts. What precautions would 
you advise for him and for his parents? Should school officials be 
aware of this condition? 

1 . Indicate to which branch(es) of the immune system the fol- 
lowing statements apply, using H for the humoral branch 
and CM for the cell-mediated branch. Some statements may 
apply to both branches. 

Involves class I MHC molecules 

Responds to viral infection 

Involves T helper cells 

Involves processed antigen 

Most likely responds following an organ 

transplant 

f. Involves T cytotoxic cells 

g. Involves B cells 

h. Involves T cells 

i. Responds to extracellular bacterial infection 

j. Involves secreted antibody 

k. Kills virus-infected self-cells 

2. Specific immunity exhibits four characteristic attributes, 
which are mediated by lymphocytes. List these four attrib- 
utes and briefly explain how they arise. 

3. Name three features of a secondary immune response that 
distinguish it from a primary immune response. 

4. Compare and contrast the four types of antigen-binding 
molecules used by the immune system — antibodies, T-cell 
receptors, class I MHC molecules, and class II MHC mole- 
cules — in terms of the following characteristics: 

a. Specificity for antigen 

b. Cellular expression 

c. Types of antigen recognized 

Go to www.whfreeman.com/immunology Self- Test 

Review and quiz of key terms 



5. Fill in the blanks in the following 
appropriate terms: 

a. , , and all function as antigen- 
presenting cells. 

b. Antigen-presenting cells deliver a signal to 

cells. 

c. Only antigen-presenting cells express class 

MHC molecules, whereas nearly all cells express class 
MHC molecules. 

d. antigens are internalized by antigen-presenting 

cells, degraded in the , and displayed with class 

MHC molecules on the cell surface. 

e. antigens are produced in altered self-cells, de- 
graded in the , and displayed with class 

MHC molecules on the cell surface. 



6. Briefly describe the three maj( 
response. 



in the inflammatory 
7. The T cell is said to be class I restricted. What does this 



. Match each term related to 
most appropriate descriptic 
scription may be used once 



mate immunity (a-p) with the 
i listed below (1-19). Each de- 
nore than once, or not at all. 



_ Fimbriae or pili 

_ Exudate 

_ Sebum 

_ Margination 

_ Dermis 

_ Lysosome 

_ Histamine 

_ Macrophage 

_ Lysozyme 

_ Bradykinin 

_ Interferon 

_ Edema 

_ Complement 

_ Extravasation 

_ C-reactive protein 

_ Phagosome 



Descriptions 

( 1 ) Thin outer layer of skin 

(2) Layer of skin containing blood vessels and sebaceous 
glands 

(3) One of several acute-phase proteins 

(4) Hydrolytic enzyme found in mucous secretions 

(5) Migration of a phagocyte through the endothelial wall 
into the tissues 

(6) Acidic antibacterial secretion found on the skin 

(7) Has antiviral activity 

(8) Induces vasodilation 

(9) Accumulation of fluid in intercellular space, resulting in 
swelling 

(10) Large vesicle containing ingested particulate material 

(11) Accumulation of dead cells, digested material, and fluid 

(12) Adherence of phagocytic cells to the endothelial wall 



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(13) Structures involved in microbial adherence ti 
membranes 

(14) Stimulates pain receptors in the skin 

(15) Phagocytic cell found in the tissues 

(16) Phagocytic cell found in the blood 

(17) Group of serum proteins involved in cell lysis and clear- 
ance of antigen 

(18) Cytoplasmic vesicle containing degradative enzymes 

(19) Protein-rich fluid that leaks from the capillaries into the 

9. Innate and adaptive immunity act in cooperative and inter- 
dependent ways to protect the host. Discuss the collabora- 
tion of these two forms of immunity. 



1 0. How might an arthropod, such as a cockroach or beetle, pro- 
tect itself from infection? In what ways might the innate im- 
mune responses of an arthropod be similar to those of a 
plant and how might they differ? 

1 1 . Give examples of mild and severe consequences of immune 
dysfunction. What is the most common cause of immunod- 
eficiency throughout the world today? 

1 2. Adaptive immunity has evolved in vertebrates but they have 
also retained innate immunity. What would be the disadvan- 
tages of having only an adaptive immune system? Comment 
on how possession of both types of immunity enhances pro- 
tection against infection. 



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Cells and Organs of the 
Immune System 



THE IMMUNE SYSTEM CONSISTS OF M, 
organs and tissues that are found throughout the 
body. These organs can be classified functionally 
into two main groups. The primary lymphoid organs provide 
appropriate microenvironments for the development and 
maturation of lymphocytes. The secondary lymphoid organs 
trap antigen from defined tissues or vascular spaces and are 
sites where mature lymphocytes can interact effectively with 
that antigen. Blood vessels and lymphatic systems connect 
these organs, uniting them into a functional whole. 

Carried within the blood and lymph and populating the 
lymphoid organs are various white blood cells, or leuko- 
cytes, that participate in the immune response. Of these 
cells, only the lymphocytes possess the attributes of diversity, 
specificity, memory, and self/nonself recognition, the hall- 
marks of an adaptive immune response. All the other cells 
play accessory roles in adaptive immunity, serving to activate 
lymphocytes, to increase the effectiveness of antigen clear- 
ance by phagocytosis, or to secrete various immune-effector 
molecules. Some leukocytes, especially T lymphocytes, se- 
crete various protein molecules called cytokines. These mol- 
ecules act as immunoregulatory hormones and play 
important roles in the regulation of immune responses. This 
chapter describes the formation of blood cells, the properties 
of the various immune-system cells, and the functions of the 
lymphoid organs. 



Hematopoiesis 

All blood cells arise from a type of cell called the hematopoi- 
etic stem cell (HSC). Stem cells are cells that can differentiate 
into other cell types; they are self-renewing — they maintain 
their population level by cell division. In humans, 
hematopoiesis, the formation and development of red and 
white blood cells, begins in the embryonic yolk sac during the 
first weeks of development. Here, yolk-sac stem cells differen- 
tiate into primitive erythroid cells that contain embryonic 
hemoglobin. In the third month of gestation, hematopoietic 
stem cells migrate from the yolk sac to the fetal liver and then 
to the spleen; these two organs have major roles in 
hematopoiesis from the third to the seventh months of gesta- 
tion. After that, the differentiation of HSCs in the bone mar- 
row becomes the major factor in hematopoiesis, and by birth 
there is little or no hematopoiesis in the liver and spleen. 

It is remarkable that every functionally specialized, ma- 
ture blood cell is derived from the same type of stem cell. In 




■ Hematopoiesis 

■ Cells of the Immune System 

■ Organs of the Immune System 

■ Systemic Function of the Immune System 

■ Lymphoid Cells and Organs — Evolutionary 
Comparisons 



contrast to a unipotent cell, which differentiates into a single 
cell type, a hematopoietic stem cell is multipotent, or pluripo- 
tent, able to differentiate in various ways and thereby generate 
erythrocytes, granulocytes, monocytes, mast cells, lympho- 
cytes, and megakaryocytes. These stem cells are few, normally 
fewer than one HSC per 5 X 10 4 cells in the bone marrow. 

The study of hematopoietic stem cells is difficult both be- 
cause of their scarcity and because they are hard to grow in 
vitro. As a result, little is known about how their proliferation 
and differentiation are regulated. By virtue of their capacity 
for self-renewal, hematopoietic stem cells are maintained at 
stable levels throughout adult life; however, when there is an 
increased demand for hematopoiesis, HSCs display an enor- 
mous proliferative capacity. This can be demonstrated in 
mice whose hematopoietic systems have been completely de- 
stroyed by a lethal dose of x-rays (950 rads; one rad repre- 
sents the absorption by an irradiated target of an amount of 
radiation corresponding to 100 ergs/gram of target). Such ir- 
radiated mice will die within 10 days unless they are infused 
with normal bone-marrow cells from a syngeneic (genetically 
identical) mouse. Although a normal mouse has 3 X 10 8 
bone-marrow cells, infusion of only 10 4 -10 5 bone-marrow 
cells (i.e., 0.01%-0.1% of the normal amount) from a donor 
is sufficient to completely restore the hematopoietic system, 



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2 25 mac79 Mac 7 9 : 4 5_ BW : Gg^sby et al . / Immunology 5e : 



which demonstrates the enormous proliferative and differ- progenitor cell (Figure 2-1). The types and amounts of 

entiative capacity of the stem cells. growth factors in the microenvironment of a particular stem 

Early in hematopoiesis, a multipotent stem cell differenti- cell or progenitor cell control its differentiation. During the 

ates along one of two pathways, giving rise to either a com- development of the lymphoid and myeloid lineages, stem 

mon lymphoid progenitor cell or a common myeloid cells differentiate into progenitor cells, which have lost the 



VISUALIZING CONCEPTS 





UK rr'Q- 





Basophil progenitor 


Platelets 


-%y 




Megakaryocyte 


Erythrocyte 


(O) 




Erythroid pr< 




Dendritic cell 



^Q Hematopoiesis. Self-renewing hematopoietic of the myeloid lineage arise from myeloid progenitors. Note that 
stem cells give rise to lymphoid and myeloid progenitors. All lym- some dendritic cells come from lymphoid progenitors, others 
phoid cells descend from lymphoid progenitor cells and all cells from myeloid precursors. 



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' Immunology 5e: 



capacity for self- renewal and are committed to a particular cell 
lineage. Common lymphoid progenitor cells give rise to B, T, 
and NK (natural killer) cells and some dendritic cells. Myeloid 
stem cells generate progenitors of red blood cells (erythro- 
cytes), many of the various white blood cells (neutrophils, 
eosinophils, basophils, monocytes, mast cells, dendritic cells), 
and platelets. Progenitor commitment depends on the acquisi- 
tion of responsiveness to particular growth factors and cy- 
tokines. When the appropriate factors and cytokines are 
present, progenitor cells proliferate and differentiate into the 
corresponding cell type, either a mature erythrocyte, a partic- 
ular type of leukocyte, or a platelet-generating cell (the 
megakaryocyte). Red and white blood cells pass into bone- 
marrow channels, from which they enter the circulation. 

In bone marrow, hematopoietic cells grow and mature on 
a meshwork of stromal cells, which are nonhematopoietic 
cells that support the growth and differentiation of hema- 
topoietic cells. Stromal cells include fat cells, endothelial cells, 
fibroblasts, and macrophages. Stromal cells influence the dif- 
ferentiation of hematopoietic stem cells by providing a 
hematopoietic-inducing microenvironment (HIM) con- 
sisting of a cellular matrix and factors that promote growth 
and differentiation. Many of these hematopoietic growth 
factors are soluble agents that arrive at their target cells by 
diffusion, others are membrane-bound molecules on the 
surface of stromal cells that require cell-to-cell contact be- 
tween the responding cells and the stromal cells. During in- 
fection, hematopoiesis is stimulated by the production of 
hematopoietic growth factors by activated macrophages and 
T cells. 

Hematopoiesis Can Be Studied In Vitro 

Cell-culture systems that can support the growth and differ- 
entiation of lymphoid and myeloid stem cells have made it 



possible to identify many hematopoietic growth factors. In 
these in vitro systems, bone-marrow stromal cells are cul- 
tured to form a layer of cells that adhere to a petri dish; 
freshly isolated bone-marrow hematopoietic cells placed on 
this layer will grow, divide, and produce large visible colonies 
(Figure 2-2). If the cells have been cultured in semisolid agar, 
their progeny will be immobilized and can be analyzed for 
cell types. Colonies that contain stem cells can be replated to 
produce mixed colonies that contain different cell types, in- 
cluding progenitor cells of different cell lineages. In contrast, 
progenitor cells, while capable of division, cannot be replated 
and produce lineage-restricted colonies. 

Various growth factors are required for the survival, pro- 
liferation, differentiation, and maturation of hematopoietic 
cells in culture. These growth factors, the hematopoietic 
cytokines, are identified by their ability to stimulate the for- 
mation of hematopoietic cell colonies in bone-marrow 
cultures. Among the cytokines detected in this way was a 
family of acidic glycoproteins, the colony-stimulating fac- 
tors (CSFs), named for their ability to induce the formation 
of distinct hematopoietic cell lines. Another important 
hematopoietic cytokine detected by this method was the gly- 
coprotein erythropoietin (EPO). Produced by the kidney, 
this cytokine induces the terminal development of erythro- 
cytes and regulates the production of red blood cells. Fur- 
ther studies showed that the ability of a given cytokine to 
signal growth and differentiation is dependent upon the 
presence of a receptor for that cytokine on the surface of the 
target cell — commitment of a progenitor cell to a particular 
differentiation pathway is associated with the expression of 
membrane receptors that are specific for particular cy- 
tokines. Many cytokines and their receptors have since been 
shown to play essential roles in hematopoiesis. This topic is 
explored much more fully in the chapter on cytokines 
(Chapter 11). 



Culture in 

semisolid agar n visible colonies of 



.^jj^^aipfe^ 



■iM 



(a) Experimental scheme for culturing hematopoietic 
cells. Adherent bone-marrow stromal cells form a matrix on which 
the hematopoietic cells proliferate. Single cells can be transferred 
to semisolid agar for colony growth and the colonies analyzed for 
differentiated cell types, (b) Scanning electron micrograph of cells 



in long-term culture of human bone marrow. [Photograph fron 
M.J. Cline and D. W. Golde, 1979, Nature 277:180; reprinted b 
permission; © 1979 Macmillan Magazines Ltd., micrograph cow 
tesy ofS. Quan.} 



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' Immunology 5e: 



Hematopoiesis Is Regulated at the 
Genetic Level 

The development of pluripotent hematopoietic stem cells 
into different cell types requires the expression of different 
sets of lineage-determining and lineage-specific genes at ap- 
propriate times and in the correct order. The proteins speci- 
fied by these genes are critical components of regulatory 
networks that direct the differentiation of the stem cell and 
its descendants. Much of what we know about the depen- 
dence of hematopoiesis on a particular gene comes from 
studies of mice in which a gene has been inactivated or 
"knocked out" by targeted disruption, which blocks the pro- 
duction of the protein that it encodes (see Targeted Disrup- 
tion of Genes, in Chapter 23). If mice fail to produce red cells 
or particular white blood cells when a gene is knocked out, 
we conclude that the protein specified by the gene is neces- 
sary for development of those cells. Knockout technology is 
one of the most powerful tools available for determining the 
roles of particular genes in a broad range of processes and it 
has made important contributions to the identification of 
many genes that regulate hematopoiesis. 

Although much remains to be done, targeted disruption 
and other approaches have identified a number of transcrip- 
tion factors (Table 2-1) that play important roles in 
hematopoiesis. Some of these transcription factors affect 
many different hematopoietic lineages, and others affect only 
a single lineage, such as the developmental pathway that leads 
to lymphocytes. One transcription factor that affects multi- 
ple lineages is GATA-2, a member of a family of transcription 
factors that recognize the tetranucleotide sequence GATA, a 
nucleotide motif in target genes. A functional GATA-2 gene, 
which specifies this transcription factor, is essential for the 
development of the lymphoid, erythroid, and myeloid lin- 
eages. As might be expected, animals in which this gene is 
disrupted die during embryonic development. In contrast to 
GATA-2, another transcription factor, Ikaros, is required 
only for the development of cells of the lymphoid lineage. Al- 
though Ikaros knockout mice do not produce significant 



Dependent lineage 



CATA-1 


Erythroid 


CATA-2 


Erythroid, myeloid, lymph 


PU.1 


Erythroid (maturational st 
stages), lymphoid 


BM11 


Myeloid, lymphoid 


Ikaros 


Lymphoid 


Oct-2 


B lymphoid (differentiatio 



numbers of B, T, and NK cells, their production of erythro- 
cytes, granulocytes, and other cells of the myeloid lineage is 
unimpaired. Ikaros knockout mice survive embryonic devel- 
opment, but they are severely compromised immunologi- 
cally and die of infections at an early age. 

Hematopoietic Homeostasis Involves 
Many Factors 

Hematopoiesis is a continuous process that generally main- 
tains a steady state in which the production of mature blood 
cells equals their loss (principally from aging). The average 
erythrocyte has a life span of 120 days before it is phagocytosed 
and digested by macrophages in the spleen. The various white 
blood cells have life spans ranging from a few days, for neu- 
trophils, to as long as 20-30 years for some T lymphocytes. To 
maintain steady-state levels, the average human being must 
produce an estimated 3.7 X 10 11 white blood cells per day. 

Hematopoiesis is regulated by complex mechanisms that 
affect all of the individual cell types. These regulatory mech- 
anisms ensure steady-state levels of the various blood cells, 
yet they have enough built-in flexibility so that production of 
blood cells can rapidly increase tenfold to twentyfold in re- 
sponse to hemorrhage or infection. Steady-state regulation of 
hematopoiesis is accomplished in various ways, which in- 
clude: 

■ Control of the levels and types of cytokines produced by 
bone-marrow stromal cells 

■ The production of cytokines with hematopoietic activity 
by other cell types, such as activated T cells and 
macrophages 

■ The regulation of the expression of receptors for 
hematopoietically active cytokines in stem cells and 
progenitor cells 



The removal of sc 
cell death 



e cells by the controlled induction of 



A failure in one or a combination of these regulatory mecha- 
nisms can have serious consequences. For example, abnormal- 
ities in the expression of hematopoietic cytokines or their 
receptors could lead to unregulated cellular proliferation and 
may contribute to the development of some leukemias. Ulti- 
mately, the number of cells in any hematopoietic lineage is set 
by a balance between the number of cells removed by cell death 
and the number that arise from division and differentiation. 
Any one or a combination of regulatory factors can affect rates 
of cell reproduction and differentiation. These factors can also 
determine whether a hematopoietic cell is induced to die. 

Programmed Cell Death Is an Essential 
Homeostatic Mechanism 

Programmed cell death, an induced and ordered process in 
which the cell actively participates in bringing about its own 
demise, is a critical factor in the homeostatic regulation of 



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many types of cell populations, including those of the 
hematopoietic system. 

Cells undergoing programmed cell death often exhibit 
distinctive morphologic changes, collectively referred to 
as apoptosis (Figures 2-3, 2-4). These changes include a 
pronounced decrease in cell volume, modification of the cy- 
toskeleton that results in membrane blebbing, a condensa- 
tion of the chromatin, and degradation of the DNA into 
smaller fragments. Following these morphologic changes, an 
apoptotic cell sheds tiny membrane-bounded apoptotic bod- 
ies containing intact organelles. Macrophages quickly phago- 
cytose apoptotic bodies and cells in the advanced stages of 
apoptosis. This ensures that their intracellular contents, in- 
cluding proteolytic and other lytic enzymes, cationic pro- 
teins, and oxidizing molecules are not released into the 
surrounding tissue. In this way, apoptosis does not induce a 
local inflammatory response. Apoptosis differs markedly 
from necrosis, the changes associated with cell death arising 
from injury. In necrosis the injured cell swells and bursts, re- 



leasing its contents and possibly triggering a damaging in- 
flammatory response. 

Each of the leukocytes produced by hematopoiesis has a 
characteristic life span and then dies by programmed cell 
death. In the adult human, for example, there are about 
5 X 10 10 neutrophils in the circulation. These cells have a 
life span of only a few days before programmed cell death 
is initiated. This death, along with constant neutrophil 
production, maintains a stable number of these cells. If 
programmed cell death fails to occur, a leukemic state may 
develop. Programmed cell death also plays a role in main- 
taining proper numbers of hematopoietic progenitor cells. 
For example, when colony-stimulating factors are re- 
moved, progenitor cells undergo apoptosis. Beyond 
hematopoiesis, apoptosis is important in such immuno- 
logical processes as tolerance and the killing of target cells 
by cytotoxic T cells or natural killer cells. Details of the 
mechanisms underlying apoptosis are emerging; Chapter 
13 describes them in detail. 



Chromatin clumping /Q' 

Swollen organelles 
Flocculent mitochondria 



a compaction 
and segregation 
Condensation of 




Nuclear fragmentat: 

- 
Apoptotic bodies 



**■ Comparison of morphologic changes that occur in 
apoptosis and necrosis. Apoptosis, which results in the programmed 
cell death of hematopoietic cells, does not induce a local inflamma- 



tory response. 


n contrast, necrosis, the process tha 


leads to dea 


of injured cells 


results in release ofthe cells' content 


, which may 


ducealocalin 


ammatory response. 





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' Immunology 5e: 



Cells and Organs of the Immune System chapter 2 29 





* 



fffA 








■ mf 


'< 


. | 


■ ' ■ ^H 1 

■ ;.- . ; ■. i« X j 






| Apoptosis. Light micrographs of (a) normal thymo- 
cytes (developing T cells in the thymus) and (b) apoptotic thymo- 
cytes. Scanning electron micrographs of (c) normal and (d) 



apoptotic thymocytes. [From B. A. Osborne and S. Smith, 1997, Jou 
nal of NIH Research 9:35; courtesy B. A. Osborne, University of Mas 
achusetts at Amherst.] 



The expression of several genes accompanies apoptosis 
in leukocytes and other cell types (Table 2-2). Some of the 
proteins specified by these genes induce apoptosis, others 
are critical during apoptosis, and still others inhibit apop- 
tosis. For example, apoptosis can be induced in thymocytes 
by radiation, but only if the protein p53 is present; many 
cell deaths are induced by signals from Fas, a molecule pre- 
sent on the surface of many cells; and proteases known as 
caspases take part in a cascade of reactions that lead to 
apoptosis. On the other hand, members of the bcl-2 (B-cell 
lymphoma 2) family of genes, bcl-2 and bcl-X L encode pro- 
tein products that inhibit apoptosis. Interestingly, the first 
member of this gene family, bcl-2, was found in studies that 
were concerned not with cell death but with the uncon- 
trolled proliferation of B cells in a type of cancer known as 
B-lymphoma. In this case, the bcl-2 gene was at the break- 
point of a chromosomal translocation in a human B-cell 
lymphoma. The translocation moved the bcl-2 gene into 
the immunoglobulin heavy-chain locus, resulting in tran- 



scriptional activation of the bcl-2 gene and overproduction 
of the encoded Bcl-2 protein by the lymphoma cells. The 
resulting high levels of Bcl-2 are thought to help transform 
lymphoid cells into cancerous lymphoma cells by inhibit- 
ing the signals that would normally induce apoptotic cell 
death. 

Bcl-2 levels have been found to play an important role in 
regulating the normal life span of various hematopoietic cell 
lineages, including lymphocytes. A normal adult has about 
5 L of blood with about 2000 lymphocytes/mm 3 for a total of 
about 10 10 lymphocytes. During acute infection, the lym- 
phocyte count increases 4- to 15-fold, giving a total lympho- 
cyte count of 40-50 X 10 9 . Because the immune system 
cannot sustain such a massive increase in cell numbers for an 
extended period, the system needs a means to eliminate un- 
needed activated lymphocytes once the antigenic threat has 
passed. Activated lymphocytes have been found to express 
lower levels of Bcl-2 and therefore are more susceptible to the 
induction of apoptotic death than are naive lymphocytes or 



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' Immunology 5e: 



30 


Introduction 




| TABLE 2-2 [ 


Gene 


Function 


Role in apoptosis 


bcl-2 


Prevents apoptosis 


Inhibits 


bax 


Opposes bcl-2 


Promotes 


bcl-X L (bcl-Long) 


Prevents apoptosis 


Inhibits 


bcl-Xs (bcl-Short) 


Opposes bd-X L 


Promotes 


caspase (several 
different ones) 


Protease 


Promotes 


fas 


induces apoptosis 


Initiates 



memory cells. However, if the lymphocytes continue to be 
activated by antigen, then the signals received during activa- 
tion block the apoptotic signal. As antigen levels subside, so 
does activation of the block and the lymphocytes begin to die 
by apoptosis (Figure 2-5). 



Hematopoietic Stem Cells Can Be Enriched 

I. L. Weissman and colleagues developed a novel way of en- 
riching the concentration of mouse hematopoietic stem cells, 
which normally constitute less than 0.05% of all bone- 
marrow cells in mice. Their approach relied on the use of an- 
tibodies specific for molecules known as differentiation 
antigens, which are expressed only by particular cell types. 
They exposed bone-marrow samples to antibodies that had 
been labeled with a fluorescent compound and were specific 
for the differentiation antigens expressed on the surface of 
mature red and white blood cells (Figure 2-6). The labeled cells 
were then removed by flow cytometry with a fluorescence- 
activated cell sorter (see Chapter 6) . After each sorting, the remain- 
ing cells were assayed to determine the number needed for 
restoration of hematopoiesis in a lethallyx- irradiated mouse. 
As the pluripotent stem cells were becoming relatively more 
numerous in the remaining population, fewer and fewer 
cells were needed to restore hematopoiesis in this system. 
Because stem cells do not express differentiation antigens 




Continued activating signals 
(e.g., cytokines, T H cells, antigen) 



| Regulation of activated B-cell numbers by apoptosis. 

Activation of B cells induces increased expression of cytokine recep- 
tors and decreased expression of Bcl-2. Because Bcl-2 prevents apop- 
tosis, its reduced level in activated B cells is an important factor in 



aking activated B cells more susceptible to programmed cell death 
an either naive or memory B cells. A reduction in activating signals 
ickly leads to destruction of excess activated B cells by apoptosis. 
nilar processes occur in T cells. 



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' Immunology 5e: 




®^o© 

U ® ® ® 



(N) 






React with 
Fl-antibodies 
to differentiation 



Fully / 










enriched / 


1 Partly 




cells / 


//enriched 
/ cells 


/ Unenriched 
/ cells 



Number of cells injected into lethally irradiated n 




J ® 



®® ®@© cr entiated 

U ®(i) ® 



Fl-antibodies 
against Sca-1 




© ® 



® 





irichment of the pluripotent stem cells from bone 
marrow, (a) Differentiated hematopoietic cells (white) are removed 
by treatment with fluorescently labeled antibodies (Fl-antibodies) 
specific for membrane molecules expressed on differentiated lin- 
eages but absent from the undifferentiated stem cells (S) and prog- 
enitor cells (P). Treatment of the resulting partly enriched preparation 
with antibody specific for Sca-1, an early differentiation antigen, re- 
moved most of the progenitor cells. M = monocyte; B = basophil 
N = neutrophil; Eo = eosinophil; L = lymphocyte; E = erythrocyte, 
(b) Enrichment of stem-cell preparations is measured by their ability 
to restore hematopoiesis in lethally irradiated mice. Only animals in 
which hematopoiesis occurs survive. Progressive enrichment ol 
stem cells is indicated by the decrease in the number of injected cells 
needed to restore hematopoiesis. A total enrichment of about 1000- 
fold is possible by this procedure. 



known to be on developing and mature hematopoietic 
cells, by removing those hematopoietic cells that express 
known differentiation antigens, these investigators were able 
to obtain a 50- to 200-fold enrichment of pluripotent stem 
cells. To further enrich the pluripotent stem cells, the re- 
maining cells were incubated with various antibodies raised 
against cells likely to be in the early stages of hematopoiesis. 
One of these antibodies recognized a differentiation antigen 
called stem-cell antigen 1 (Sca-1). Treatment with this anti- 
body aided capture of undifferentiated stem cells and yielded 
a preparation so enriched in pluripotent stem cells that an 
aliquot containing only 30-100 cells routinely restored 
hematopoiesis in a lethally x-irradiated mouse, whereas 



more than 10 nonenriched bone-marrow cells were needed 
for restoration. Using a variation of this approach, H. 
Nakauchi and his colleagues have devised procedures that al- 
low them to show that, in 1 out of 5 lethally irradiated mice, 
a single hematopoietic cell can give rise to both myeloid and 
lymphoid lineages (Table 2-3). 

It has been found that CD34, a marker found on about 1% 
of hematopoietic cells, while not actually unique to stem 
cells, is found on a small population of cells that contains 
stem cells. By exploiting the association of this marker with 
stem cell populations, it has become possible to routinely en- 
rich preparations of human stem cells. The administration of 
human-cell populations suitably enriched for CD34 + cells 



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TABLE 2-3 



Number of 
enriched HSCs 



reconstituted (%) 

9 of 41 (21.9%) 
5 of 21 (23.8%) 

9 of 1 7 (52.9%) 

10 of 11 (90.9%) 
4 of 4 (100%) 



(the "+" indicates that the factor is present on the cell mem- 
brane) can reconstitute a patient's entire hematopoietic sys- 
tem (see Clinical Focus). 

A major tool in studies to identify and characterize the 
human hematopoietic stem cell is the use of SCID (severe 
combined immunodeficiency) mice as in vivo assay systems 
for the presence and function of HSCs. SCID mice do not 



have B and T lymphocyt' 

tion of foreign cells, tissi 
animals do not reject tr 
containing HSCs or tiss 
row. It is necessary to use 
or alternative hosts in 
there is no hun 



:s and are unable to mount adaptive 
is those that act in the normal rejec- 
es, and organs. Consequently, these 
msplanted human cell populations 
les such as thymus and bone mar- 
immunodeficient mice as surrogate 
luman stem-cell research because 



s no human equivalent of the irradiated mouse. SCID 
mplanted with fragments of human thymus and bone 
;upport the differentiation of human hematopoietic 
stem cells into mature hematopoietic cells. Different subpop- 
ulations of CD34 human bone-marrow cells are injected 
into these SCID-human mice, and the development of vari- 
ous lineages of human cells in the bone-marrow fragment is 
subsequently assessed. In the absence of human growth fac- 
tors, only low numbers of granulocyte-macrophage progeni- 
tors develop. However, when appropriate cytokines such as 
erythropoietin and others are administered along with 
CD34 + cells, progenitor and mature cells of the myeloid, 
lymphoid, and erythroid lineages develop. This system has 
enabled the study of subpopulations of CD34 cells and the 
effect of human growth factors on the differentiation of var- 
ious hematopoietic lineages. 



Cells ofthe Immune System 

Lymphocytes are the central cells of the immune system, re- 
sponsible for adaptive immunity and the immunologic at- 
tributes of diversity, specificity, memory, and self/nonself 
recognition. The other types of white blood cells play impor- 



tant roles, engulfing and destroying microorganisms, pre- 
senting antigens, and secreting cytokines. 

Lymphoid Cells 

Lymphocytes constitute 20%-40% ofthe body's white blood 
cells and 99% ofthe cells in the lymph (Table 2-4). There are 
approximately 10 11 (range depending on body size and age: 
~10 10 -10 12 ) lymphocytes in the human body. These lym- 
phocytes continually circulate in the blood and lymph and 
are capable of migrating into the tissue spaces and lymphoid 
organs, thereby integrating the immune system to a high 
degree. 

The lymphocytes can be broadly subdivided into three 
populations — B cells, T cells, and natural killer cells — on the 
basis of function and cell-membrane components. Natural 
killer cells (NK cells) are large, granular lymphocytes that do 
not express the set of surface markers typical of B or T cells. 
Resting B and T lymphocytes are small, motile, nonphago- 
cytic cells, which cannot be distinguished morphologically. B 
and T lymphocytes that have not interacted with antigen — 
referred to as naive, or unprimed — are resting cells in the G 
phase of the cell cycle. Known as small lymphocytes, these 
cells are only about 6 |jim in diameter; their cytoplasm forms 
a barely discernible rim around the nucleus. Small lympho- 
cytes have densely packed chromatin, few mitochondria, and 
a poorly developed endoplasmic reticulum and Golgi appa- 
ratus. The naive lymphocyte is generally thought to have a 
short life span. Interaction of small lymphocytes with anti- 
gen, in the presence of certain cytokines discussed later, in- 
duces these cells to enter the cell cycle by progressing from G 
into Gi and subsequently into S, G 2 , and M (Figure 2-7a). As 
they progress through the cell cycle, lymphocytes enlarge 
into 15 |jLm-diameter blast cells, called lymphoblasts; these 
cells have a higher cytoplasm:nucleus ratio and more or- 
ganellar complexity than small lymphocytes (Figure 2-7b). 

Lymphoblasts proliferate and eventually differentiate into 
effector cells or into memory cells. Effector cells function in 
s ways to eliminate antigen. These cells have short life 



Cell type Cells/mm 3 % 

Red blood cells 5.0 X 10 6 

Platelets 2.5 X 10 5 

Leukocytes 7.3 X 10 3 

Neutrophil 50-70 

Lymphocyte 20-40 

Monocyte 1-6 



Basophil 



www.whfreeman.com/immunology i , .j| A 

and Organs ofthe Immune System 



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• 33 mac79 Mac 7 9 : 4 5_ BW : Gg^sby et al . / Immunology 5e : 



I! lymphocyte 




(gene activation) 






fpJi^ 


^■©iV. 


\tifi 


V /I 



Blast cell (T or B) 
1 5 |Xm diameter 




Plasma cell (B) 
1 5 |Jm diameter 



activated small lymphocytes, (a) A 
ned) lymphocyte resides in the C 
cycle. At this stage, B and T lymphocytes cannot be 
distinguished morphologically. After antigen activation, a B or T cell 
enters the cell cycle and enlarges into a lymphoblast, which under- 
goes several rounds of cell division and, eventually, generates effector 
cells and memory cells. Shown here are cells of the B-cell lineage, 
(b) Electron micrographs of a small lymphocyte (left) showing con- 



densed chro 
phoblast (cei 
cell (right) si 



» of a 



:ell, an enlarged lym- 
r) showing decondensed chromatin, and a plasma 
*ing abundant endoplasmic reticulum arranged in 
;s and a prominent nucleus that has been pushed to 
a characteristically eccentric position. The three cells are shown at 
different magnifications. [Micrographs courtesy of Dr. J. R. Goodman, 
Dept. of Pediatrics, University of California at San Francisco.] 



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' Immunology 5e: 



CLINICAL FOCUS 



Stem Cells — Clinical Uses 
and Potential 



transplanta- 
tion holds great promise for the regener- 
ation of diseased, damaged, or defective 
tissue. Hematopoietic stem cells are al- 
ready used to restore hematopoietic 
cells, and their use is described in the 
clinic below. However, rapid advances in 
stem-cell research have raised the possi- 
bility that other stem-cell types, too, may 
soon be routinely employed for replace- 
ment of other cells and tissues. Two 
properties of stem cells underlie their 
utility and promise. They have the capac- 
ity to give rise to more differentiated 
cells, and they are self-renewing, because 
each division of a stem cell creates at 
least one stem cell. If stem cells are clas- 
sified according to their descent and de- 
velopmental potential, four levels of 
stem cells can be recognized: totipotent, 
pluripotent, multipotent, and unipotent. 
Totipotent cells can give rise to an en- 
tire organism. A fertilized egg, the zygote, 
is a totipotent cell. In humans the initial di- 
visions of the zygote and its descendants 
produce cells that are also totipotent. In 
fact, identical twins, each with its own pla- 
centa, develop when totipotent cells sepa- 
rate and develop into genetically identical 
fetuses. Pluripotent stem cells arise from 
totipotent cells and can give rise to most 
but not all of the cell types necessary for fe- 
tal development. For example, human 
pluripotent stem cells can give rise to all of 
the cells of the body but cannot generate a 
placenta. Further differentiation of pluripo- 
tent stem cells leads to the formation of 
multipotent and unipotent stem cells. 
Multipotent stem cells can give rise to only 
a limited number of cell types, and unipo- 
tent cells to a single cell type. Pluripotent 
cells, called embryonic stem cells, or sim- 
ply ES cells, can be isolated from early em- 
bryos, and for many years it has been 
possible to grow mouse ES cells as cell 



lines in the laboratory. Strikingly, these ES 
cells can be induced to generate many dif- 
ferent types of cells. Mouse ES cells have 
been shown to give rise to muscle cells, 
nerve cells, liver cells, pancreatic cells, and, 
of course, hematopoietic cells. 

Recent advances have made it possible 
to grow lines of human pluripotent cells. 
This is a development of considerable im- 
portance to the understanding of human 
development, and it has great therapeutic 
potential. In vitro studies of the factors that 
determine or influence the development of 
human pluripotent stem cells along one de- 
velopmental path as opposed to another 
will provide considerable insight into the 
factors that affect the differentiation of cells 
into specialized types. There is also great in- 
terest in exploring the use of pluripotent 



stem cells to generate cells and tissues that 
could be used to replace diseased or dam- 
aged ones. Success in this endeavor would 
be a major advance because transplanta- 
tion medicine now depends totally upon do- 
nated organs and tissues. Unfortunately, 
the need far exceeds the number of dona- 
tions and is increasing. Success in deriving 
practical quantities of cells, tissues, and or- 
gans from pluripotent stem cells would pro- 
vide skin replacement for burn patients, 
heart muscle cells for those with chronic 
heart disease, pancreatic islet cells for pa- 
tients with diabetes, and neurons for use in 
Parkinson's disease or Alzheimer's disease. 
The transplantation of hematopoietic 
stem cells (HSCs) is an important ther- 
apy for patients whose hematopoietic 
systems must be replaced. It has three 
major applications: 



1. Providii 
genetic 



afunc 



lly determim 
(deficiency, s 





Human pluripotent sterr 
some of which are show 
http://www.nih. gov/ n< 

Biophoto Associates/Scie 
Researchers; AFIP/Scienc 
Michler /Science Photo Library/Photo Researchers.] 



muscle cells Pancreatic islet cells 



cells can differentiate into a variety of different cell types 
here. [Adapted from Stem Cells: A Primer, NIH web sitt 
vs/stemcell/primer.ritm. Micrographs (left to right): 
ce Source/Photo Researchers; Biophoto Associates/Phot 
Source/Photo Researchers; Astrid e[ Hanns-Frieder 



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: al . / Immunology 5e : 



combined immunodeficiency 
(SCID). 
2. Replacing a defective hematopoietic 
system with a functional one to cure 
some patients who have a life- 
threatening nonmalignant genetic 
disorder in hematopoiesis, such as 



ickle- 



>r thai 



3. Restoring the hematopoiel 
of cancer patients after tr 
with doses of chemotherapeutic 
agents and radiation so high that 
they destroy the system. These 
high-dose regimens can be much 
more effective at killing tumor cells 
than are therapies that use more 
conventional doses of cytotoxic 
agents. Stem-cell transplantation 
makes it possible to recover from 
such drastic treatment. Also, certain 

acute myeloid leukemia, can be 
cured only by destroying the source 
of the leukemia cells, the patient's 
own hematopoietic system. 
Restoration of the hematopoietic sys- 
tem by transplanting stem cells is facili- 
tated by several important technical 
considerations. First, HSCs have extraordi- 
nary powers of regeneration. Experiments 
in mice indicate that only a few — perhaps, 
on occasion, a single HSC-can com- 
pletely restore the erythroid population and 
the immune system. In humans it is neces- 
sary to administer as little as 10% of a 
donor's total volume of bone marrow to 
provide enough HSCs to completely re- 
store the hematopoietic system. Once in- 
jected into a vein, HSCs enter the 
circulation and find their own way to the 
bone marrow, where they begin the process 
of engraftment. There is no need for a sur- 
geon to directly inject the cells into bones. 
In addition, HSCs can be preserved by 
freezing. This means that hematopoietic 
cells can be "banked." After collection, the 
cells are treated with a cryopreservative, 
frozen, and then stored for later use. When 
needed, the frozen preparation is thawed 
and infused into the patient, where it re- 
constitutes the hematopoietic system. This 
cell-freezing technology even makes it pos- 



sible for individuals to store their own 
hematopoietic cells for transplantation to 
themselves at a later time. Currently, this 
procedure is used to allow cancer patients 
to donate cells before undergoing chemo- 
therapy and radiation treatments and then 
to reconstitute their hematopoietic system 
from their own stem cells. Hematopoietic 
stem cells are found in cell populations that 
display distinctive surface antigens. One of 
these antigens is CD34, which is present on 
only a small percentage (-1%) of the cells 
in adult bone marrow. An antibody specific 
for CD34 is used to select cells displaying 
this antigen, producing a population en- 
riched in CD34 + stem cells. Various ver- 
sions ofthis selection procedure have been 
used to enrich populations of stem cells 
from a variety of sources. 

Transplantation of stem cell popula- 
tions may be autologous (the recipient is 
also the donor), syngeneic (the donor is 
genetically identical, i.e., an identical twin 
of the recipient), or allogeneic (the donor 
and recipient are not genetically identical). 
In any transplantation procedure, genetic 
differences between donor and recipient 
can lead to immune-based rejection reac- 
tions. Aside from host rejection of trans- 
planted tissue (host versus graft), 
lymphocytes in the graft can attack the re- 
cipient's tissues, thereby causing graft- 
versus-host disease (CVHD), a life- 
threatening affliction. In order to suppress 
rejection reactions, powerful immunosup- 
pressive drugs must be used. Unfortu- 
nately, these drugs have serious side 
effects, and immunosuppression in- 
creases the patient's risk of infection and 
further growth of tumors. Consequently, 
HSC transplantation has fewest complica- 
tions when there is genetic identity be- 
tween donor and recipient. 

At one time, bone-marrow transplanta- 
tion was the only way to restore the 
hematopoietic system. However, the essen- 
tial element of bone-marrow transplanta- 
tion is really stem-cell transplantation. 
Fortunately, significant numbers of stem 
cells can be obtained from other tissues, 
such as peripheral blood and umbilical-cord 
blood ("cord blood"). These alternative 
sources of HSCs are attractive because the 



donor does not have to undergo anesthesia 
and the subsequent highly invasive proce- 
dure that extracts bone marrow. Many in the 
transplantation community believe that pe- 
ripheral blood will replace marrow as the 
major source of hematopoietic stem cells 
for many applications. To obtain HSC-en- 
riched preparations from peripheral blood, 
agents are used to induce increased num- 
bers of circulating HSCs, and then the HSC- 
containing fraction is separated from the 
plasma and red blood cells in a process 
called leukopheresis. If necessary, further 
purification can be done to remove T cells 
and to enrich the CD34 + population. 

Umbilical cord blood already contains a 
significant number of hematopoietic stem 
cells. Furthermore, it is obtained from pla- 
cental tissue (the "afterbirth") which is nor- 
mally discarded. Consequently, umbilical 
cord blood has become an attractive 
source of cells for HSC transplantation. Al- 
though HSCs from cord blood fail to en- 
graft somewhat more often than do cells 
from peripheral blood, grafts of cord blood 
cells produce CVHD less frequently than 
do marrow grafts, probably because cord 
blood has fewer mature T cells. 

Beyond its current applications in can- 
cer treatment, many researchers feel that 
autologous stem-cell transplantation will 
be useful for gene therapy, the introduction 
of a normal gene to correct a disorder 
caused by a defective gene. Rapid ad- 
vances in genetic engineering may soon 
make gene therapy a realistic treatment for 
genetic disorders of blood cells, and 
hematopoietic stem cells are attractive ve- 
hicles for such an approach. The therapy 
would entail removing a sample of 
hematopoietic stem cells from a patient, 
inserting a functional gene to compensate 
for the defective one, and then reinjecting 
the engineered stem cells into the donor. 
The advantage of using stem cells in gene 
therapy is that they are self renewing. Con- 
sequently, at least in theory, patients would 
have to receive only a single injection of en- 
gineered stem cells. In contrast, gene ther- 
apy with engineered mature lymphocytes 
or other blood cells would require periodic 
injections because these cells are not ca- 
pable of self renewal. 



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' Immunology 5e: 



spans, generally ranging from a few days to a few weeks. 
Plasma cells — the antibody-secreting effector cells of the B- 
cell lineage — have a characteristic cytoplasm that contains 
abundant endoplasmic reticulum (to support their high rate 
of protein synthesis) arranged in concentric layers and also 
many Golgi vesicles (see Figure 2-7). The effector cells of the 
T-cell lineage include the cytokine-secreting T helper cell 
(T H cell) and the T cytotoxic lymphocyte (T c cell). Some of 
the progeny of B and T lymphoblasts differentiate into mem- 
ory cells. The persistence of this population of cells is respon- 
sible for life-long immunity to many pathogens. Memory 
cells look like small lymphocytes but can be distinguished 
from naive cells by the presence or absence of certain cell- 
membrane molecules. 

Different lineages or maturational stages of lymphocytes 
can be distinguished by their expression of membrane mole- 
cules recognized by particular monoclonal antibodies (anti- 
bodies that are specific for a single epitope of an antigen; see 
Chapter 4 for a description of monoclonal antibodies). All of 
the monoclonal antibodies that react with a particular mem- 
brane molecule are grouped together as a cluster of dif- 
ferentiation (CD). Each new monoclonal antibody that 
recognizes a leukocyte membrane molecule is analyzed for 
whether it falls within a recognized CD designation; if it does 



not, it is given a new CD designation reflecting a new mem- 
brane molecule. Although the CD nomenclature was origi- 
nally developed for the membrane molecules of human 
leukocytes, the homologous membrane molecules of other 
species, such as mice, are commonly referred to by the same 
CD designations. Table 2-5 lists some common CD mole- 
cules (often referred to as CD markers) found on human 
lymphocytes. However, this is only a partial listing of the 
more than 200 CD markers that have been described. A com- 
plete list and description of known CD markers is in the ap- 
pendix at the end of this book. 

The general characteristics and functions of B and T lym- 
phocytes were described in Chapter 1 and are reviewed 
briefly in the next sections. These central cells of the immune 
system will be examined in more detail in later chapters. 

B LYMPHOCYTES 

The B lymphocyte derived its letter designation from its site 
of maturation, in the bursa of Fabricius in birds; the name 
turned out to be apt, for bone marrow is its major site of mat- 
uration in a number of mammalian species, including hu- 
mans and mice. Mature B cells are definitively distinguished 
from other lymphocytes by their synthesis and display of 
membrane-bound immunoglobulin (antibody) molecules, 



CD2 
CD3 



in molecule; signal transductio 
ransduction element of T-cell 



CD16 (Fc-yRIII) 

CD21 (CR2) 

CD28 

CD32 (Fc-yRII) 
CD35 (CR1) 

CD40 
CD45 
CD56 
"Synonyms are show 



Adhesion molecule that binds to clas 
MHC molecules; signal transductioi 

Unknown 

Adhesion molecule that binds to clas 
MHC molecules; signal transductioi 
Low-affinity receptor for Fc region of I 

Receptor for complement (C3d) and 
Epstein-Barr virus 

Receptor for co-stimulatory B7 molec 

on antigen-presenting cells 
Receptor for Fc region of IgG 
Receptor for complement (C3b) 
Signal transduction 

Adhesion molecule 



(usually) (usually) 

(subset) 

(usually) (usually) (variable) 



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' Immunology 5e: 



which serve as receptors for antigen. Each of the approxi- 
mately 1.5 X 10 5 molecules of antibody on the membrane of 
a single B cell has an identical binding site for antigen. 
Among the other molecules expressed on the membrane of 
mature B cells are the following: 

■ B220 (a form of CD45) is frequently used as a marker 
for B cells and their precursors. However, unlike 
antibody it is not expressed uniquely by B-lineage cells. 

■ Class II MHC molecules permit the B cell to function as 
an antigen-presenting cell (APC). 

. CR1 (CD35) and CR2 (CD21) are receptors for certain 
complement products. 

■ FcyRII (CD32) is a receptor for IgG, a type of antibody. 

■ B7-1 (CD80) and B7-2 (CD86) are molecules that 
interact with CD28 and CTLA-4, important regulatory 
molecules on the surface of different types of T cells, 
including T H cells. 

■ CD40 is a molecule that interacts with CD40 ligand on 
the surface of helper T cells. In most cases this 
interaction is critical for the survival of antigen- 
stimulated B cells and for their development into 
antibody-secreting plasma cells or memory B cells. 

Interaction between antigen and the membrane-bound anti- 
body on a mature naive B cell, as well as interactions with T 
cells and macrophages, selectively induces the activation and 
differentiation of B-cell clones of corresponding specificity. 
In this process, the B cell divides repeatedly and differentiates 
over a 4- to 5-day period, generating a population of plasma 
cells and memory cells. Plasma cells, which have lower levels 
of membrane-bound antibody than B cells, synthesize and 
secrete antibody. All clonal progeny from a given B cell se- 
crete antibody molecules with the same antigen-binding 
specificity. Plasma cells are terminally differentiated cells, 
and many die in 1 or 2 weeks. 

T LYMPHOCYTES 

T lymphocytes derive their name from their site of matura- 
tion in the thymus. Like B lymphocytes, these cells have 
membrane receptors for antigen. Although the antigen- 
binding T-cell receptor is structurally distinct from im- 
munoglobulin, it does share some common structural 
features with the immunoglobulin molecule, most notably in 
the structure of its antigen-binding site. Unlike the mem- 
brane-bound antibody on B cells, though, the T-cell receptor 
(TCR) does not recognize free antigen. Instead the TCR rec- 
ognizes only antigen that is bound to particular classes of 
self- molecules. Most T cells recognize antigen only when it is 
bound to a self-molecule encoded by genes within the major 
histocompatibility complex (MHC). Thus, as explained in 
Chapter 1, a fundamental difference between the humoral 
and cell-mediated branches of the immune system is that the 
B cell is capable of binding soluble antigen, whereas the T cell 



MHCn 



gen boi 
CD4ve 



is restricted to binding antigen displayed on self-cells. To be 
recognized by most T cells, this antigen must be displayed to- 
gether with MHC molecules on the surface of antigen-pre- 
senting cells or on virus-infected cells, cancer cells, and 
grafts. The T-cell system has developed to eliminate these al- 
tered self-cells, which pose a threat to the normal functioning 
of the body. 

Like B cells, T cells express distinctive membrane mole- 
cules. All T-cell subpopulations express the T-cell receptor, a 
complex of polypeptides that includes CD3; and most can be 
distinguished by the presence of one or the other of two 
membrane molecules, CD4 and CD8. In addition, most ma- 
ture T cells express the following membrane molecules: 

■ CD28, a receptor for the co-stimulatory B7 family of 
molecules present on B cells and other antigen- 
presenting cells 

■ CD45, a signal-transduction molecule 

T cells that express the membrane glycoprotein molecule 
CD4 are restricted to recognizing antigen bound to class II 
olecules, whereas T cells expressing CD8, a dimeric 
ne glycoprotein, are restricted to recognition of anti- 
nd to class I MHC molecules. Thus the expression of 
sus CD8 corresponds to the MHC restriction of the 
T cell. In general, expression of CD4 and of CD8 also defines 
two major functional subpopulations of T lymphocytes. 
CD4 + T cells generally function as T helper (T H ) cells and are 
class-II restricted; CD8 + T cells generally function as T cyto- 
toxic (T c ) cells and are class-I restricted. Thus the ratio of T H 
to T c cells in a sample can be approximated by assaying the 
number of CD4 and CD8 T cells. This ratio is approxi- 
mately 2: 1 in normal human peripheral blood, but it may be 
significantly altered by immunodeficiency diseases, autoim- 
mune diseases, and other disorders. 

The classification of CD4 class II-restricted cells as T H 
cells and CD8 class I-restricted cells as T c cells is not ab- 
solute. Some CD4 + cells can act as killer cells. Also, some T c 
cells have been shown to secrete a variety of cytokines and ex- 
ert an effect on other cells comparable to that exerted by T H 
cells. The distinction between T H and T c cells, then, is not al- 
ways clear; there can be ambiguous functional activities. 
However, because these ambiguities are the exception and 
not the rule, the generalization of T helper (T H ) cells as being 
CD4 + and class-II restricted and of T cytotoxic cells (T c ) as 
being CD8 and class-I restricted is assumed throughout 
this text, unless otherwise specified. 

T H cells are activated by recognition of a 
MHC complex on an antigen-presenting 
tion, the T H cell begins to divide and gives 
effector cells, each specific for the same 
MHC complex. These T H cells secrete 
which play a central role in 
and other cells that participate 

Changes in the pattern of cytokines produced by T H cells 
i response that develops ami 



antigen-class II 
11. After activa- 
ise to a clone of 
ntigen-class II 

ytokines, 
cells, T cells, 

response. 



change the type of 



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' Immunology 5e: 



other leukocytes. The T H 1 response produces a cytokine 
profile that supports inflammation and activates mainly cer- 
tain T cells and macrophages, whereas the T H 2 response ac- 
tivates mainly B cells and immune responses that depend 
upon antibodies. T c cells are activated when they interact 
with an antigen-class I MHC complex on the surface of an 
altered self-cell (e.g., a virus-infected cell or a tumor cell) in 
the presence of appropriate cytokines. This activation, which 
results in proliferation, causes the T c cell to differentiate into 
an effector cell called a cytotoxic T lymphocyte (CTL). In 
contrast to T H cells, most CTLs secrete few cytokines. In- 
stead, CTLs acquire the ability to recognize and eliminate al- 
tered self-cells. 

Another subpopulation of T lymphocytes — called T sup- 
pressor (T s ) cells — has been postulated. It is clear that some 
T cells help to suppress the humoral and the cell-mediated 
branches of the immune system, but the actual isolation and 
cloning of normal T s cells is a matter of controversy and dis- 
pute among immunologists. For this reason, it is uncertain 
whether T s cells do indeed constitute a separate functional 
subpopulation of T cells. Some immunologists believe that 
the suppression mediated by T cells observed in some sys- 
tems is simply the consequence of activities of T H or T c sub- 
populations whose end results are suppressive. 

NATURAL KILLER CELLS 

The natural killer cell was first described in 1976, when it was 
shown that the body contains a small population of large, 
granular lymphocytes that display cytotoxic activity against a 
wide range of tumor cells in the absence of any previous im- 
munization with the tumor. NK cells were subsequently 
shown to play an important role in host defense both against 
tumor cells and against cells infected with some, though not 
all, viruses. These cells, which constitute 5%-10% of lym- 
phocytes in human peripheral blood, do not express the 
membrane molecules and receptors that distinguish T- and 
B-cell lineages. Although NK cells do not have T-cell recep- 
tors or immunoglobulin incorporated in their plasma mem- 
branes, they can recognize potential target cells in two 
different ways. In some cases, an NK cell employs NK cell re- 
ceptors to distinguish abnormalities, notably a reduction in 
the display of class I MHC molecules and the unusual profile 
of surface antigens displayed by some tumor cells and cells 
infected by some viruses. Another way in which NK cells rec- 
ognize potential target cells depends upon the fact that some 
tumor cells and cells infected by certain viruses display anti- 
gens against which the immune system has made an anti- 
body response, so that antitumor or antiviral antibodies are 
bound to their surfaces. Because NK cells express CD16, a 
membrane receptor for the carboxyl-terminal end of the IgG 
molecule, called the Fc region, they can attach to these anti- 
bodies and subsequently destroy the targeted cells. This is an 
example of a process known as antibody-dependent cell- 
mediated cytotoxicity (ADCC). The exact mechanism of 
NK-cell cytotoxicity, the focus of much current experimental 
study, is described further in Chapter 14. 



Several observations suggest that NK cells play an impor- 
tant role in host defense against tumors. For example, in hu- 
mans the Chediak-Higashi syndrome — an autosomal 
recessive disorder — is associated with impairment in neu- 
trophils, macrophages, and NK cells and an increased inci- 
dence of lymphomas. Likewise, mice with an autosomal 
mutation called beige lack NK cells; these mutants are more 
susceptible than normal mice to tumor growth following in- 
jection with live tumor cells. 

There has been growing recognition of a cell type, the 
NK1-T cell, that has some of the characteristics of both T 
cells and NK cells. Like T cells, NK1-T cells have T cell recep- 
tors (TCRs). Unlike most T cells, the TCRs of NK1-T cells in- 
teract with MHC-like molecules called CD1 rather than with 
class I or class II MHC molecules. Like NK cells, they have 
variable levels of CD 16 and other receptors typical of NK 
cells, and they can kill cells. A population of triggered NK1-T 
of the cytokines 
by B cells as well as 
expansion of cyto- 
w this cell type as 



cells can rapidly secrete large 

needed to support antibody prodi 

inflammation and the developme 

toxic T cells. Some immunologi 

a kind of rapid response system 

vide early help while conventional T H responses 

developing. 



) pro 
e still 



Mononuclear Phagocytes 

The mononuclear phagocytic system consists of monocytes 
circulating in the blood and macrophages in the tissues 
(Figure 2-8). During hematopoiesis in the bone marrow, 
granulocyte-monocyte progenitor cells differentiate into 
promonocytes, which leave the bone marrow and enter 
the blood, where they further differentiate into mature 
monocytes. Monocytes circulate in the bloodstream for 
about 8 h, during which they enlarge; they then migrate into 
the tissues and differentiate into specific tissue macrophages 
or, as discussed later, into dendritic cells. 

Differentiation of a monocyte into a tissue macrophage 
involves a number of changes: The cell enlarges five- to ten- 
fold; its intracellular organelles increase in both number and 
complexity; and it acquires increased phagocytic ability, pro- 
duces higher levels of hydrolytic enzymes, and begins to se- 
crete a variety of soluble factors. Macrophages are dispersed 
throughout the body. Some take up residence in particular 
tissues, becoming fixed macrophages, whereas others remain 
motile and are called free, or wandering, macrophages. Free 
macrophages travel by amoeboid movement throughout 
the tissues. Macrophage-like cells serve different functions in 
different tissues and are named according to their tissue 
location: 

■ Alveolar macrophages in the lung 

■ Histiocytes in connective tissues 

■ Kupffer cells in the liver 

■ Mesangial cells in the kidney 



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Phagolysosome 



^| Typical morphology 
macrophage. Macrophages are five- to ti 



monocyte 
argerthanmo 



organelles, especially ly: 

■ Microglial cells in the brain 

■ Osteoclasts in bone 

Although normally in a resting state, macrophages ; 
vated by a variety of stimuli in the course of £ 
sponse. Phagocytosis of particulate antigens serves as an 
initial activating stimulus. However, macrophage activity can 
be further enhanced by cytokines secreted by activated T H 
cells, by mediators of the inflammatory response, and by 
components of bacterial cell walls. One of the most potent 
activators of macrophages is interferon gamma (IFN-y) se- 
creted by activated T H cells. 

Activated macrophages are more effective than resting 
ones in eliminating potential pathogens, because they exhibit 
greater phagocytic activity, an increased ability to kill in- 
gested microbes, increased secretion of inflammatory medi- 
ators, and an increased ability to activate T cells. In addition, 



activated macrophages, but not resting ones, s 
cytotoxic proteins that help them eliminate a broad range of 
pathogens, including virus- infected cells, tumor cells, and in- 
tracellular bacteria. Activated macrophages also express 
higher levels of class II MHC molecules, allowing them to 
function more effectively as antigen-presenting cells. Thus, 
macrophages and T H cells facilitate each other's 
during the immune response. 



PHAGOCYTOSIS 

Macrophages are capable of ingesting and digesting exoge- 
nous antigens, such as whole microorganisms and insoluble 
particles, and endogenous matter, such as injured or dead 
host cells, cellular debris, and activated clotting factors. In the 
first step in phagocytosis, macrophages are attracted by and 
move toward a variety of substances generated in an immune 
response; this process is called chemotaxis. The next step in 
phagocytosis is adherence of the antigen to the macrophage 
cell membrane. Complex antigens, such as whole bacterial 
cells or viral particles, tend to adhere well and are readily 
phagocytosed; isolated proteins and encapsulated bacteria 
tend to adhere poorly and are less readily phagocytosed. Ad- 
herence induces membrane protrusions, called pseudopo- 
dia, to extend around the attached material (Figure 2-9a). 
Fusion of the pseudopodia encloses the material within a 
membrane-bounded structure called a phagosome, which 
then enters the endocytic processing pathway (Figure 2-9b). 
In this pathway, a phagosome moves toward the cell interior, 
where it fuses with a lysosome to form a phagolysosome. 
Lysosomes contain lysozyme and a variety of other hy- 
drolytic enzymes that digest the ingested material. The di- 
gested contents of the phagolysosome are then eliminated in 
a process called exocytosis (see Figure 2-9b). 

The macrophage membrane has receptors for certain 
classes of antibody. If an antigen (e.g., a bacterium) is coated 
with the appropriate antibody, the complex of antigen and 
antibody binds to antibody receptors on the macrophage 
membrane more readily than antigen alone and phagocyto- 
sis is enhanced. In one study, for example, the rate of phago- 
cytosis of an antigen was 4000-fold higher in the presence of 
specific antibody to the antigen than in its absence. Thus, an- 
tibody functions as an opsonin, a molecule that binds to 
both antigen and macrophage and enhances phagocytosis. 
The process by which particulate antigens are rendered more 
susceptible to phagocytosis is called opsonization. 

ANTIMICROBIAL AND CYTOTOXIC ACTIVITIES 
A number of antimicrobial and cytotoxic substances pro- 
duced by activated macrophages can destroy phagocytosed 
microorganisms (Table 2-6). Many of the mediators of cyto- 
toxicity listed in Table 2-6 are reactive forms of oxygen. 

OXYGEN-DEPENDENT KILLING MECHANISMS Activated 
phagocytes produce a number of reactive oxygen intermedi- 
ates (ROIs) and reactive nitrogen intermediates that have 



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Antigenic peptide/class II MHC 



Exocytosed degraded material 



| Macrophages can ingest and degrade particulate 
antigens, including bacteria, (a) Scanning electron micrograph of a 
macrophage. Note the long pseudopodia extending toward and mak- 
ing contact with bacterial cells, an early step in phagocytosis, (b) 
Phagocytosis and processing of exogenous antigen by macrophages. 



Most of the products resulting from digestion of ingested material 
are exocytosed, but some peptide products may interact with class II 
MHC molecules, forming complexes that move to the cell surface, 
where they are presented to T H cells. [Photograph by L Nilsson, © 
Boehringer Ingelheim International GmbH.] 



potent antimicrobial activity. During phagocytosis, a meta- 
bolic process known as the respiratory burst occurs in acti- 
vated macrophages. This process results in the activation of a 
membrane-bound oxidase that catalyzes the reduction of 
oxygen to superoxide anion, a reactive oxygen intermediate 
that is extremely toxic to ingested microorganisms. The su- 
peroxide anion also generates other powerful oxidizing 
agents, including hydroxyl radicals and hydrogen peroxide. 
As the lysosome fuses with the phagosome, the activity of 
myeloperoxidase produces hypochlorite from hydrogen per- 



Oxygen-dependent killing 



Oxygen-independent killing 



Reactive oxygen intermediates 
0' 2 (superoxide anion) 
OH" (hydroxyl radicals) 
H 2 2 (hydrogen peroxide) 
CIO" (hypochlorite anion) 

Reactive nitrogen intermediates 
NO (nitric oxide) 
N0 2 (nitrogen dioxide) 
HN0 2 (nitrous acid) 

Others 

NH 2 CL (monochloramine) 



Defensins 

Tumor necrosis factor < 

(macrophage only) 
Lysozyme 
Hydrolytic enzymes 



oxide and chloride ions. Hypochlorite, the active agent of 
household bleach, is toxic to ingested microbes. When 
macrophages are activated with bacterial cell-wall compo- 
nents such as lipopolysaccharide (LPS) or, in the case of my- 
cobacteria, muramyl dipeptide (MDP), together with a 
T-cell-derived cytokine (IFN-7), they begin to express high 
levels of nitric oxide synthetase (NOS), an enzyme that oxi- 
dizes L-arginine to yield L-citrulline and nitric oxide (NO), a 



L-arginine + 2 +NADPH > 

NO + L-citrulline + NADP 

Nitric oxide has potent antimicrobial activity; it also can 
combine with the superoxide anion to yield even more po- 
tent antimicrobial substances. Recent evidence suggests that 
much of the antimicrobial activity of macrophages against 
bacteria, fungi, parasitic worms, and protozoa is due to nitric 
oxide and substances derived from it. 

OXYGEN-INDEPENDENT KILLING MECHANISMS Acti- 
vated macrophages also synthesize lysozyme and various hy- 
drolytic enzymes whose degradative activities do not require 
oxygen. In addition, activated macrophages produce a group 
of antimicrobial and cytotoxic peptides, commonly known 
as defensins. These molecules are cysteine-rich cationic pep- 
tides containing 29-35 amino-acid residues. Each peptide, 
which contains six invariant cysteines, forms a circular mole- 
cule that is stabilized by intramolecular disulfide bonds. 
These circularized defensin peptides have been shown to 
form ion-permeable channels in bacterial cell membranes. 
Defensins can kill a variety of bacteria, including Staphylo- 
coccus aureus, Streptococcus pneumoniae, Escherichia coli, 



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Pseudomonas aeruginosa, and Haemophilus influenzae. Acti- 
vated macrophages also secrete tumor necrosis factor a 
(TNF-a), a cytokine that has a variety of effects and is cyto- 
toxic for some tumor cells. 

ANTIGEN PROCESSING AND PRESENTATION 
Although most of the antigen ingested by macrophages is de- 
graded and eliminated, experiments with radiolabeled anti- 
gens have demonstrated the presence of antigen peptides on 
the macrophage membrane. As depicted in Figure 2-9b, 
phagocytosed antigen is digested within the endocytic pro- 
cessing pathway into peptides that associate with class II 
MHC molecules; these peptide-class II MHC complexes 
then move to the macrophage membrane. Activation of 
macrophages induces increased expression of both class II 
MHC molecules and the co-stimulatory B7 family of mem- 
brane molecules, thereby rendering the macrophages more 
effective in activating T H cells. This processing and presenta- 
tion of antigen, examined in detail in Chapter 7, are critical to 
T H -cell activation, a central event in the development of both 
humoral and cell-mediated immune responses. 

SECRETION OF FACTORS 

A number of important proteins central to development of 
immune responses are secreted by activated macrophages 
(Table 2-7). These include a collection of cytokines, such as 
interleukin 1 (IL-1), TNF-a and interleukin 6 (IL-6), that 
promote inflammatory responses. Typically, each of these 
agents has a variety of effects. For example, IL-1 activates 
lymphocytes; and IL- 1 , IL-6, and TNF-a promote fever by af- 
fecting the thermoregulatory center in the hypothalamus. 



TABLE 2-7 



Interleukin 1 (IL-1) 


Promotes infla 
and fever 


mmatory responses 


Interleukin 6 (IL-6) 1 


Promote innat 


e immunity and 


TNF-a J 


elimination c 


f pathogens 


Complement proteins 


Promote infla 


nmatory response 
on of pathogens 


Hydrolytic enzymes 


Promote infla 


nmatory response 


Interferon alpha 
(IFN-a) 


Activates cellu 
in the produ 
confer an an 


ar genes, resulting 
tion of proteins that 
iviral state on the cell 


Tumor necrosis factor 


Kills tumor ce 


s 


(TNF-a) 






GM-CSF 1 






G-CSF 


Promote indu 


ible hematopoiesis 


M-CSF J 







creted when the cells 
enzymes within the tissues c 
response and can, in some c; 
sue damage. Activated macrc 
tors, such as TNF-a, that c 



Activated macrophages secrete a variety of factors in- 
volved in the development of an inflammatory response. The 
complement proteins are a group of proteins that assist in 
eliminating foreign pathogens and in promoting the ensuing 
inflammatory reaction. The major site of synthesis of com- 
plement proteins is the liver, although these proteins are also 
produced in macrophages. The hydrolytic enzymes con- 
tained within the lysosomes of macrophages also can be se- 
ctivated. The buildup of these 
ontributes to the inflammatory 
ises, contribute to extensive tis- 
jphages also secrete soluble fac- 
an kill a variety of cells. The 
secretion of these cytotoxic factors has been shown to con- 
tribute to tumor destruction by macrophages. Finally, as 
mentioned earlier, activated macrophages secrete a number 
of cytokines that stimulate inducible hematopoiesis. 

Granulocytic Cells 

The granulocytes are classified as neutrophils, eosinophils, 
or basophils on the basis of cellular morphology and cyto- 
plasmic staining characteristics (Figure 2-10). The neu- 
trophil has a multilobed nucleus and a granulated cytoplasm 
that stains with both acid and basic dyes; it is often called a 
polymorphonuclear leukocyte (PMN) for its multilobed nu- 
cleus. The eosinophil has a bilobed nucleus and a granulated 
cytoplasm that stains with the acid dye eosin red (hence its 
name). The basophil has a lobed nucleus and heavily granu- 
lated cytoplasm that stains with the basic dye methylene blue. 
Both neutrophils and eosinophils are phagocytic, whereas 
basophils are not. Neutrophils, which constitute 50%-70% 
of the circulating white blood cells, are much more numer- 
ous than eosinophils (l%-3%) or basophils (<1%). 

NEUTROPHILS 

Neutrophils are produced by hematopoiesis in the bone mar- 
row. They are released into the peripheral blood and circulate 
for 7-10 h before migrating into the tissues, where they have 
a life span of only a few days. In response to many types of in- 
fections, the bone marrow releases more than the usual num- 
ber of neutrophils and these cells generally are the first to 
arrive at a site of inflammation. The resulting transient in- 
crease in the number of circulating neutrophils, called leuko- 
cytosis, is used medically as an indication of infection. 

Movement of circulating neutrophils into tissues, called 
extravasation, takes several steps: the cell first adheres to 
the vascular endothelium, then penetrates the gap between 
adjacent endothelial cells lining the vessel wall, and finally 
penetrates the vascular basement membrane, moving out 
into the tissue spaces. (This process is described in detail in 
Chapter 15.) A number of substances generated in an inflam- 
matory reaction serve as chemotactic factors that promote 
accumulation of neutrophils at an inflammatory site. Among 
these chemotactic factors are some of the complement 



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' Immunology 5e: 




owing typical morphology of gra 
cytes. Note differences in the shape of the nucleus and in the 
ber and shape of cytoplasmic granules. 



components, components of the blood-clotting system, and sev- 
eral cytokines secreted by activated T H cells and macrophages. 

Like macrophages, neutrophils are active phagocytic cells. 
Phagocytosis by neutrophils is similar to that described for 
macrophages, except that the lytic enzymes and bactericidal 
substances in neutrophils are contained within primary and 
secondary granules (see Figure 2- 10a). The larger, denser pri- 
mary granules are a type of lysosome containing peroxidase, 
lysozyme, and various hydrolytic enzymes. The smaller sec- 
ondary granules contain collagenase, lactoferrin, and lyso- 
zyme. Both primary and secondary granules fuse with 
phagosomes, whose contents are then digested and elimi- 
nated much as they are in macrophages. 



Neutrophils also employ both oxygen-dependent and 
oxygen-independent pathways to generate antimicrobial 
substances. Neutrophils are in fact much more likely than 
macrophages to kill ingested microorganisms. Neutrophils 
exhibit a larger respiratory burst than macrophages and con- 
sequently are able to generate more reactive oxygen interme- 
diates and reactive nitrogen intermediates (see Table 2-6). In 
addition, neutrophils express higher levels of defensins than 
macrophages do. 

EOSINOPHILS 

Eosinophils, like neutrophils, are motile phagocytic cells that 
can migrate from the blood into the tissue spaces. Their 
phagocytic role is significantly less important than that of 
neutrophils, and it is thought that they play a role in the de- 
fense against parasitic organisms (see Chapter 17). The se- 
creted contents of eosinophilic granules may damage the 
parasite membrane. 

BASOPHILS 

Basophils are nonphagocytic granulocytes that function by 
releasing pharmacologically active substances from their cy- 
toplasmic granules. These substances play a major role in cer- 
tain allergic responses. 

MAST CELLS 

Mast-cell precursors, which are formed in the bone marrow 
by hematopoiesis, are released into the blood as undifferenti- 
ated cells; they do not differentiate until they leave the blood 
and enter the tissues. Mast cells can be found in a wide vari- 
ety of tissues, including the skin, connective tissues of various 
organs, and mucosal epithelial tissue of the respiratory, geni- 
tourinary, and digestive tracts. Like circulating basophils, 
these cells have large numbers of cytoplasmic granules that 
contain histamine and other pharmacologically active sub- 
stances. Mast cells, together with blood basophils, play an im- 
portant role in the development of allergies. 

DENDRITIC CELLS 

The dendritic cell (DC) acquired its name because it is cov- 
ered with long membrane extensions that resemble the den- 
drites of nerve cells. Dendritic cells can be difficult to isolate 
because the conventional procedures for cell isolation tend to 
damage their long extensions. The development of isolation 
techniques that employ enzymes and gentler dispersion has 
facilitated isolation of these cells for study in vitro. There are 
many types of dendritic cells, although most mature den- 
dritic cells have the same major function, the presentation of 
antigen to T H cells. Four types of dendritic cells are known: 
Langerhans cells, interstitial dendritic cells, myeloid cells, 
and lymphoid dendritic cells. Each arises from hematopoi- 
etic stem cells via different pathways and in different loca- 
tions. Figure 2-11 shows that they descend through both the 
myeloid and lymphoid lineages. Despite their differences, 



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tibody c 
antigen 



mplexes. The interaction of B cells with this bound 
in have important effects on B cell responses. 



© 

Common myeloid 
progenitor 



Common lymphoid 




| Dendritic cells arise from both the myeloid and lym- 
phoid lineages. The myeloid pathway that gives rise to the mono- 
cyte/macrophage cell type also gives rise to dendritic cells. Some 
dendritic cells also arise from the lymphoid lineage. These consider- 
ations do not apply to follicular dendritic cells, which are not derived 
from bone marrow. 



they all constitutively express high levels of both class II 
MHC molecules and members of the co-stimulatory B7 fam- 
ily. For this reason, they are more potent antigen-presenting 
cells than macrophages and B cells, both of which need to be 
activated before they can function as antigen-presenting cells 
(APCs). Immature or precursor forms of each of these types 
of dendritic cells acquire antigen by phagocytosis or endocy- 
tosis; the antigen is processed, and mature dendritic cells pre- 
sent it to T H cells. Following microbial invasion or during 
inflammation, mature and immature forms of Langerhans 
cells and interstitial dendritic cells migrate into draining 
lymph nodes, where they make the critical presentation of 
antigen to T H cells that is required for the initiation of re- 
sponses by those key cells. 

Another type of dendritic cell, the follicular dendritic cell 
(Figure 2-12), does not arise in bone marrow and has a dif- 
ferent function from the antigen-presenting dendritic cells 
described above. Follicular dendritic cells do not express class 
II MHC molecules and therefore do not function as antigen- 
presenting cells for T H -cell activation. These dendritic cells 
were named for their exclusive location in organized struc- 
tures of the lymph node called lymph follicles, which are rich 
in B cells. Although they do not express class II molecules, 
follicular dendritic cells express high levels of membrane re- 
ceptors for antibody, which allows the binding of antigen-an- 



Organs ofthe Immune System 

A number of morphologically and functionally diverse or- 
gans and tissues have various functions in the development 
of immune responses. These can be distinguished by func- 
tion as the primary and secondary lymphoid organs (Fig- 
ure 2-13). The thymus and bone marrow are the primary (or 
central) lymphoid organs, where maturation of lymphocytes 
takes place. The lymph nodes, spleen, and various mucosal- 
associated lymphoid tissues (MALT) such as gut-associated 
lymphoid tissue (GALT) are the secondary (or peripheral) 
lymphoid organs, which trap antigen and provide sites for 
mature lymphocytes to interact with that antigen. In addi- 
tion, tertiary lymphoid tissues, which normally contain 
fewer lymphoid cells than secondary lymphoid organs, can 
import lymphoid cells during an inflammatory response. 
Most prominent of these are cutaneous-associated lymphoid 
tissues. Once mature lymphocytes have been generated in the 
primary lymphoid organs, they circulate in the blood and 
lymphatic system, a network of vessels that collect fluid that 
has escaped into the tissues from capillaries ofthe circulatory 
system and ultimately return it to the blood. 



Primary Lymphoid Organs 



e lymphocytes generated in hematopoiesis mature 
and become committed to a particular antigenic specificity 
within the primary lymphoid organs. Only after a lympho- 




**"' Scanning electron micrograph of follicular dendritic 
cells showing long, beaded dendrites. The beads are coated with anti- 
gen-antibody complexes. The dendrites emanate from the cell body. 
[From A. K. Szakal et al., 1985, J. Immunol. 134:1353; © 1996 by 
American Association oflmmunologists, reprinted with permission.] 

Go to www.whfreeman.com/immunology i .j Animat 

Cells and Organs ofthe Immune System 



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Adenoids 

Thoracic duct 
Left subclavian 







Q| The human lymphoid system. The primary organs 
(bone marrow and thymus) are shown in red; secondary organs and 
tissues, in blue. These structurally and functionally diverse lymphoid 

shown) and lymphatic vessels (purple) through which lymphocytes 

row and thus are part of the lymphoid system. [Adapted from H. 
Lodishetal., 1995, Molecular Cell Biology, 3rd ed., Scientific American 
Books.] 



cyte has matured within a primary lymphoid organ is the cell 
immunocompetent (capable of mounting an immune re- 
sponse). T cells arise in the thymus, and in many 
mammals — humans and mice for example — B cells origi- 



THYMUS 

The thymus is the site of T-cell development and maturation. 
It is a flat, bilobed organ situated above the heart. Each lobe 
is surrounded by a capsule and is divided into lobules, which 
are separated from each other by strands of connective tissue 
called trabeculae. Each lobule is organized into two compart- 
ments: the outer compartment, or cortex, is densely packed 
with immature T cells, called thymocytes, whereas the inner 
compartment, or medulla, is sparsely populated with thymo- 
cytes. 

Both the cortex and medulla of the thymus are criss- 
crossed by a three-dimensional stromal-cell network com- 
posed of epithelial cells, dendritic cells, and macrophages, 
which make up the framework of the organ and contribute to 
the growth and maturation of thymocytes. Many of these 
stromal cells interact physically with the developing thymo- 
cytes (Figure 2-14). Some thymic epithelial cells in the outer 
cortex, called nurse cells, have long membrane extensions 
that surround as many as 50 thymocytes, forming large mul- 
ticellular complexes. Other cortical epithelial cells have long 
interconnecting cytoplasmic extensions that form a network 
and have been shown to interact with numerous thymocytes 
as they traverse the cortex. 

The function of the thymus is to generate and select a 
repertoire of T cells that will protect the body from infection. 
As thymocytes develop, an enormous diversity of T-cell re- 
ceptors is generated by a random process (see Chapter 9) that 
produces some T cells with receptors capable of recognizing 
antigen-MHC complexes. However, most of the T-cell recep- 
tors produced by this random process are incapable of recog- 
nizing antigen-MHC complexes and a small portion react 
with combinations of self antigen-MHC complexes. Using 
mechanisms that are discussed in Chapter 10, the thymus in- 
duces the death of those T cells that cannot recognize anti- 
gen-MHC complexes and those that react with self-antigen- 
MHC and pose a danger of causing autoimmune disease. 
More than 95% of all thymocytes die by apoptosis in the thy- 
mus without ever reaching maturity. 

THE THYMUS AND IMMUNE FUNCTION The role of the 
thymus in immune function can be studied in mice by exam- 
ining the effects of neonatal thymectomy, a procedure in 
which the thymus is surgically removed from newborn mice. 
These thymectomized mice show a dramatic decrease in cir- 
culating lymphocytes of the T-cell lineage and an absence of 
cell-mediated immunity. Other evidence of the importance 
of the thymus comes from studies of a congenital birth defect 
in humans (DiGeorge's syndrome) and in certain mice 
(nude mice) in which the thymus fails to develop. In both 
cases, there is an absence of circulating T cells and of cell-me- 
diated immunity and an increase in infectious disease. 

Aging is accompanied by a decline in thymic function. 
This decline may play some role in the decline in immune 
function during aging in humans and mice. The thymus 
reaches its maximal size at puberty and then atrophies, with 
a significant decrease in both cortical and medullary cells and 



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Dividing thymocyte 




Blood vessel 



Interdigitating dendritic cell 
epithelial cell 



| Diagrammatic cross section of a portion of the thy- 
mus, showing several lobules separated by connective tissue strands 
(trabeculae). The densely populated outer cortex is thought to con- 
tain many immature thymocytes (blue), which undergo rapid prolif- 
eration coupled with an enormous rate of cell death. Also present in 
the outer cortex are thymic nurse cells (gray), which are specialized 
epithelial cells with long membrane extensions that surround as 
many as 50 thymocytes. The medulla is sparsely populated and is 
thought to contain thymocytes that are more mature. During their 



stay within the thymus, thymocytes interact with various stromal 
cells, including cortical epithelial cells (light red), medullary epithelial 
cells (tan), interdigitating dendritic cells (purple), and macrophages 
(yellow). These cells produce thymic hormones and express high lev- 
els of class I and class II MHC molecules. Hassalls corpuscles, 
found in the medulla, contain concentric layers of degenerating ep- 
ithelial cells. [Adapted, with permission, from W. van Ewijk, 1991, 
Annu. Rev. Immunol. 9:591, © 1991 by Annual Reviews.] 



an increase in the total fat content of the organ. Whereas the 
average weight of the thymus is 70 g in infants, its age-depen- 
dent involution leaves an organ with an average weight of 
only 3 g in the elderly (Figure 2-15). 

A number of experiments have been designed to look at 
the effect of age on the immune function of the thymus. In 
one experiment, the thymus from a 1 -day-old or 33-month- 
old mouse was grafted into thymectomized adults. (For most 
laboratory mice, 33 months is very old.) Mice receiving the 
newborn thymus graft showed a significantly larger improve- 
ment in immune function than mice receiving the 33- 
month-old thymus. 

BONE MARROW 

In humans and mice, bone marrow is the site of B-cell origin 
and development. Arising from lymphoid progenitors, im- 
mature B cells proliferate and differentiate within the bone 
marrow, and stromal cells within the bone marrow interact 
directly with the B cells and secrete various cytokines that are 
required for development. Like thymic selection during T- 
cell maturation, a selection process within the bone n 
eliminates B cells with self-reactive antibody receptor 
process is explained in detail in Chapter 11. Bone r 
is not the site of B-cell development in all species. In birds, 
a lymphoid organ called the bursa of Fabricius, a lymphoid 



tissue associated with the gut, is the primary site of B-cell 
maturation. In mammals such as primates and rodents, there 
is no bursa and no single counterpart to it as a primary lym- 
phoid organ. In cattle and sheep, the primary lymphoid tis- 
sue hosting the maturation, proliferation, and diversification 
of B cells early in gestation is the fetal spleen. Later in gesta- 
tion, this function is assumed by a patch of tissue embedded 




Changes in the thymus with age. The thymus 
id cellularity after puberty. 



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in the wall of the intestine called the ileal Peyer's patch, which 
contains a large number (>10 10 ) B cells. The rabbit, too, uses 
gut-associated tissues such as the appendix as primary lym- 
phoid tissue for important steps in the proliferation and di- 
versification of B cells. 

Lymphatic System 

As blood circulates under pressure, its fluid component 
(plasma) seeps through the thin wall of the capillaries into 
the surrounding tissue. Much of this fluid, called interstitial 
fluid, returns to the blood through the capillary membranes. 
The remainder of the interstitial fluid, now called lymph, 
flows from the spaces in connective tissue into a network of 
tiny open lymphatic capillaries and then into a series of pro- 





Tissue space Lymphatic 


f 


\ n [> capillaries 


o 

3 


acts 




k \\ \ KrW$) 1 Lymphatic 












\ \ \ 1 1 / /^^^~^^ Lymphoid 




\k \I^-V| ( I follicle 








/ V! n 




J \q( I (^-^^ Afferent 




1 i 




Ka ^^oJ^^^r~V 1 vessel 




/ x^un^M^^, ts 




btm,;C v. # v\ 








i\*/^-"--r; . ". ' "•' 




■ . ..•' -'v- <•)■;». " ■■ l.Mliph 




'.'.•,--,'■"-''" '•■ nock- 








\^^te^/^^»7 




^ 11 ®\\ \vSqC^/\ Secondary 




Efferent «f/n\ \ follicle 




lymphatic- — __ . 1] e Germinal 











Lymphatic vessels. Small lymphatic capillaries open- 
ing into the tissue spaces pick up interstitial tissue fluid and carry it 
into progressively larger lymphatic vessels, which carry the fluid, now 
called lymph, into regional lymph nodes. As lymph leaves the nodes, 
it is carried through larger efferent lymphatic vessels, which eventu- 
ally drain into the circulatory system at the thoracic duct or right 
lymph duct (see Figure 2-13). 



gressively larger collecting vessels called lymphatic vessels 
(Figure 2-16). 

The largest lymphatic vessel, the thoracic duct, empties 
into the left subclavian vein near the heart (see Figure 2-13). 
In this way, the lymphatic system captures fluid lost from the 
blood and returns it to the blood, thus ensuring steady-state 
levels of fluid within the circulatory system. The heart does 
not pump the lymph through the lymphatic system; instead 
the flow of lymph is achieved as the lymph vessels are 
squeezed by movements of the body's muscles. A series of 
one-way valves along the lymphatic vessels ensures that 
lymph flows only in one direction. 

When a foreign antigen gains entrance to the tissues, it 
is picked up by the lymphatic system (which drains all the 
tissues of the body) and is carried to various organized 
lymphoid tissues such as lymph nodes, which trap the 
foreign antigen. As lymph passes from the tissues to lym- 
phatic vessels, it becomes progressively enriched in lympho- 
cytes. Thus, the lymphatic system also serves as a means 
of transporting lymphocytes and antigen from the connec- 
tive tissues to organized lymphoid tissues where the lympho- 
cytes may interact with the trapped antigen and undergo 



Secondary Lymphoid Organs 

Various types of organized lymphoid tissues are located 
along the vessels of the lymphatic system. Some lymphoid 
tissue in the lung and lamina propria of the intestinal wall 
consists of diffuse collections of lymphocytes and macro- 
phages. Other lymphoid tissue is organized into structures 
called lymphoid follicles, which consist of aggregates of lym- 
phoid and nonlymphoid cells surrounded by a network of 
draining lymphatic capillaries. Until it is activated by anti- 
gen, a lymphoid follicle — called a primary follicle — com- 
prises a network of follicular dendritic cells and small resting 
B cells. After an antigenic challenge, a primary follicle be- 
comes a larger secondary follicle — a ring of concentrically 
packed B lymphocytes surrounding a center (the germinal 
center) in which one finds a focus of proliferating B lympho- 
cytes and an area that contains nondividing B cells, and some 
helper T cells interspersed with macrophages and follicular 
dendritic cells (Figure 2-17). 

Most antigen-activated B cells divide and differentiate 
into antibody-producing plasma cells in lymphoid follicles, 
but only a few B cells in the antigen-activated population find 
their way into germinal centers. Those that do undergo one 
or more rounds of cell division, during which the genes that 
encode their antibodies mutate at an unusually high rate. 
Following the period of division and mutation, there is a rig- 
orous selection process in which more than 90% of these B 
cells die by apoptosis. In general, those B cells producing an- 
tibodies that bind antigen more strongly have a much better 
chance of surviving than do their weaker companions. The 
small number of B cells that survive the germinal center's rig- 
orous selection differentiate into plasma cells or memory 



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:condary lymphoid follicle consisting of a large 
germinal center (gc) surrounded by a dense mantle (m) of small lym- 
phocytes. [From W. Bloom and D. W. Fawcett, 1975, Textbook of His- 
tology, 10th ed., © 1975 by W. B. Saunders Co.] 



cells and emerge. The process of B-cell proliferation, muta- 
tion, and selection in germinal centers is described more fully 
in Chapter 11. 

Lymph nodes and the spleen are the most highly orga- 
nized of the secondary lymphoid organs; they comprise not 
only lymphoid follicles, but additional distinct regions of T- 
cell and B-cell activity, and they are surrounded by a fibrous 
capsule. Less-organized lymphoid tissue, collectively called 
mucosal-associated lymphoid tissue (MALT), is found in 
various body sites. MALT includes Peyer's patches (in the 
small intestine), the tonsils, and the appendix, as well as nu- 
merous lymphoid follicles within the lamina propria of the 
intestines and in the mucous membranes lining the upper 
airways, bronchi, and genital tract. 

LYMPH NODES 

Lymph nodes are the sites where immune responses are 
mounted to antigens in lymph. They are encapsulated bean- 
shaped structures containing a reticular network packed 
with lymphocytes, macrophages, and dendritic cells. Clus- 
tered at junctions of the lymphatic vessels, lymph nodes are 



the first organized lymphoid structure tc 
that enter the tissue spaces. As lymph percolates through a 
node, any particulate antigen that is brought in with the 
lymph will be trapped by the cellular network of phagocytic 
cells and dendritic cells (follicular and interdigitating). The 
overall architecture of a lymph node supports an ideal mi- 
croenvironment for lymphocytes to effectively encounter 
and respond to trapped antigens. 

Morphologically, a lymph node can be divided into three 
roughly concentric regions: the cortex, the paracortex, and 
the medulla, each of which supports a 
ment (Figure 2-18). The outermost layer, the cortex, c 
lymphocytes (mostly B cells), macro-phages, and follicular 
dendritic cells arranged in primary follicles. After antigenic 
challenge, the primary follicles enlarge into secondary folli- 
cles, each containing a germinal center. In children with B-cell 
deficiencies, the cortex lacks primary follicles and germinal 
centers. Beneath the cortex is the paracortex, which is popu- 
lated largely by T lymphocytes and also contains interdigitat- 
ing dendritic cells thought to have migrated from tissues to 
the node. These interdigitating dendritic cells express high 
levels of class II MHC molecules, which are necessary for pre- 
senting antigen to T H cells. Lymph nodes taken from neona- 
tally thymectomized mice have unusually few cells in the 
paracortical region; the paracortex is therefore sometimes re- 
ferred to as a thymus-dependent area in contrast to the cor- 
tex, which is a thymus-independent area. The innermost 
layer of a lymph node, the medulla, is more sparsely popu- 
lated with lymphoid-lineage cells; of those present, many are 
plasma cells actively secreting antibody molecules. 

As antigen is carried into a regional node by the lymph, it 
is trapped, processed, and presented together with class II 
MHC molecules by interdigitating dendritic cells in the para- 
cortex, resulting in the activation of T H cells. The initial acti- 
vation of B cells is also thought to take place within the 
T-cell-rich paracortex. Once activated, T H and B cells form 
small foci consisting largely of proliferating B cells at the 
edges of the paracortex. Some B cells within the foci differen- 
tiate into plasma cells secreting IgM and IgG. These foci 
reach maximum size within 4-6 days of antigen challenge. 
Within 4-7 days of antigen challenge, a few B cells and T H 
cells migrate to the primary follicles of the cortex. It is not 
known what causes this migration. Within a primary follicle, 
cellular interactions between follicular dendritic cells, B cells, 
and T H cells take place, leading to development of a sec- 
ondary follicle with a central germinal center. Some of the 
plasma cells generated in the germinal center move to the 
medullary areas of the lymph node, and many migrate to 

Afferent lymphatic vessels pierce the capsule of a lymph 
node at numerous sites and empty lymph into the subcapsu- 
lar sinus (see Figure 2- 18b). Lymph coming from the tissues 
percolates slowly inward through the cortex, paracortex, and 
medulla, allowing phagocytic cells and dendritic cells to trap 
any bacteria or particulate material (e.g., antigen-antibody 
complexes) carried by the lymph. After infection or the 



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^^ Structure of a lymph node, (a) The three layers of a 
lymph node support distinct microenvironments. (b) The left side 
depicts the arrangement of reticulum and lymphocytes within the 
various regions of a lymph node. Macrophages and dendritic cells, 
which trap antigen, are present in the cortex and paracortex. T H cells 
are concentrated in the paracortex; B cells are located primarily in the 
cortex, within follicles and germinal centers. The medulla is popu- 



lated largely by antibody-producing plasma cells. Lymphocytes circu- 
lating in the lymph are carried into the node by afferent lymphatic 
vessels; they either enter the reticular matrix of the node or pass 
through it and leave by the efferent lymphatic vessel. The right side 
of (b) depicts the lymphatic artery and vein and the postcapillary 
venules. Lymphocytes in the circulation can pass into the node from 
the postcapillary venules by a process called extravasation (inset). 



introduction of other antigens into the body, the lymph leav- 
ing a node through its single efferent lymphatic vessel is en- 
riched with antibodies newly secreted by medullary plasma 
cells and also has a fiftyfold higher concentration of lympho- 
cytes than the afferent lymph. 

The increase in lymphocytes in lymph leaving a node is 
due in part to lymphocyte proliferation within the node in 
response to antigen. Most of the increase, however, repre- 
sents blood-borne lymphocytes that migrate into the node 
by passing between specialized endothelial cells that line the 



postcapillary venules of the node. Estimates are that 25% of 
the lymphocytes leaving a lymph node have migrated across 
this endothelial layer and entered the node from the blood. 
Because antigenic stimulation within a node can increase this 
migration tenfold, the concentration of lymphocytes in a 
node that is actively responding can increase greatly, and the 
node swells visibly. Factors released in lymph nodes during 
antigen stimulation are thought to facilitate this increased 
migration. 



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: al . / Immunology 5e : 



SPLEEN 

The spleen plays a major role in mounting immune re- 
sponses to antigens in the blood stream. It is a large, ovoid 
secondary lymphoid organ situated high in the left abdomi- 
nal cavity. While lymph nodes are specialized for trapping 
antigen from local tissues, the spleen specializes in filtering 
blood and trapping blood-borne antigens; thus, it can re- 
spond to systemic infections. Unlike the lymph nodes, the 
spleen is not supplied by lymphatic vessels. Instead, blood- 
borne antigens and lymphocytes are carried into the spleen 
through the splenic artery. Experiments with radioactively 
labeled lymphocytes show that more recirculating lympho- 
cytes pass daily through the spleen than through all the 
lymph nodes combined. 



The spleen is surrounded by a capsule that extends a num- 
ber of projections (trabeculae) into the interior to form a 
compartmentalized structure. The compartments are of two 
types, the red pulp and white pulp, which are separated by a 
diffuse marginal zone (Figure 2-19). The splenic red pulp 
consists of a network of sinusoids populated by macrophages 
and numerous red blood cells (erythrocytes) and few lym- 
phocytes; it is the site where old and defective red blood cells 
are destroyed and removed. Many of the macrophages within 
the red pulp contain engulfed red blood cells or iron pigments 
from degraded hemoglobin. The splenic white pulp sur- 
rounds the branches of the splenic artery, forming a periarte- 
riolar lymphoid sheath (PALS) populated mainly by T 
lymphocytes. Primary lymphoid follicles are attached to the 



Gastric surface 




' Structure of the spleen, (a) The spleen, which is 
about 5 inches long in adults, is the largest secondary lymphoid or- 
gan. It is specialized for trapping blood-borne antigens, (b) Diagram- 
matic cross section of the spleen. The splenic artery pierces the 
capsule and divides into progressively smaller arterioles, ending in 
vascular sinusoids that drain back into the splenic vein. The erythro- 



cyte-filled red pulp surrounds the sinusoids. The white pulp forms a 
sleeve, the periarteriolar lymphoid sheath (PALS), around the arteri- 
oles; this sheath contains numerous T cells. Closely associated with 
the PALS is the marginal zone, an area rich in B cells that contains 
lymphoid follicles that can develop into secondary follicles contain- 



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' Immunology 5e: 



PALS. These follicles are rich in B cells and some of them con- 
tain germinal centers. The marginal zone, located peripheral 
to the PALS, is populated by lymphocytes and macrophages. 
Blood-borne antigens and lymphocytes enter the splee 



s trapped by interdigi- 
to the PALS. Lympho- 
the marginal zone and 



mgh the splenic artery, which t 
zone. In the marginal zone, antigen 
tating dendritic cells, which carry i 
cytes in the blood also enter sinuses 
migrate to the PALS. 

The initial activation of B and T cells takes place in the T- 
cell-rich PALS. Here interdigitating dendritic cells capture 
antigen and present it combined with class II MHC mole- 
cules to T H cells. Once activated, these T H cells can then acti- 
vate B cells. The activated B cells, together with some T H cells, 
then migrate to primary follicles in the marginal zone. Upon 
antigenic challenge, these primary follicles develop into char- 
acteristic secondary follicles containing germinal centers 
(like those in the lymph nodes), where rapidly dividing B 
cells (centroblasts) and plasma cells are surrounded by dense 
clusters of concentrically arranged lymphocytes. 

The effects of splenectomy on the immune response de- 
pends on the age at which the spleen is removed. In children, 
splenectomy often leads to an increased incidence of bacterial 
sepsis caused primarily by Streptococcus pneumoniae, Neisse- 
ria meningitidis, and Haemophilus influenzae. Splenectomy in 
adults has less adverse effects, although it leads to some in- 
crease in blood-borne bacterial infections (bacteremia). 

MUCOSAL-ASSOCIATED LYMPHOID TISSUE 

branes lining the digestive, respiratory, and 
s have a combined surface area of about 



400 m 2 (nearly the size of a basketball court) and are the ma- 
jor sites of entry for most pathogens. These vulnerable mem- 
brane surfaces are defended by a group of organized 
lymphoid tissues mentioned earlier and known collectively 
as mucosal-associated lymphoid tissue (MALT). Struc- 
turally, these tissues range from loose, barely organized clus- 
ters of lymphoid cells in the lamina propria of intestinal villi 
to well-organized structures such as the familiar tonsils and 
appendix, as well as Peyer's patches, which are found within 
the submucosal layer of the intestinal lining. The functional 
importance of MALT in the body's defense is attested to by its 
large population of antibody-producing plasma cells, whose 
number far exceeds that of plasma cells in the spleen, lymph 
nodes, and bone marrow combined. 

The tonsils are found in three locations: lingual at the 
base of the tongue; palatine at the sides of the back of the 
mouth; and pharyngeal (adenoids) in the roof of the na- 
sopharynx (Figure 2-20). All three tonsil groups are nodular 
structures consisting of a meshwork of reticular cells and 
fibers interspersed with lymphocytes, macrophages, granulo- 
cytes, and mast cells. The B cells are organized into follicles 
and germinal centers; the latter are surrounded by regions 
showing T-cell activity. The tonsils defend against antigens 
entering through the nasal and oral epithelial routes. 

The best studied of the mucous membranes is the one that 
lines the gastrointestinal tract. This tissue, like that of the res- 
piratory and urogenital tracts, has the capacity to endocytose 
antigen from the lumen. Immune reactions are initiated 
against pathogens and antibody can be generated and ex- 
ported to the lumen to combat the invading organisms. As 
shown in Figures 2-21 and 2-22, lymphoid cells are found in 
ons within this tissue. The outer mucosal epithe- 




n of tongue at lingual tonsil 



1 Three types of tonsils, (a) The position and internal J. Klein, 1982, Immunology, The Science of Self-Nonself Disc 
features of the palatine and lingual tonsils; (b) a view of the position tion, © 1982 by John Wiley and Sons, Inc.] 

of the nasopharyngeal tonsils (adenoids). [Part b adapted from 



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Intestinal lumen 




id diffuse follicles. 



lial layer contains so-called intraepithelial lymphocytes 
(IELs). Many of these lymphocytes are T cells that express 
unusual receptors (y8T-cell receptors, or y8 TCRs), which 
exhibit limited diversity for antigen. Although this popula- 
tion of T cells is well situated to encounter antigens that en- 
ter through the intestinal mucous epithelium, their actual 



function remains largely unknown The lamina propria, 
which lies under the epithelial layer, contains large numbers 
of B cells, plasma cells, activated T H cells, and macrophages 
in loose clusters. Histologic sections have revealed more than 
15,000 lymphoid follicles within the intestinal lamina pro- 
pria of a healthy child. The submucosal layer beneath the 
lamina propria contains Peyer's patches, nodules of 30-40 
lymphoid follicles. Like lymphoid follicles in other sites, 
those that compose Peyer's patches can develop into sec- 
ondary follicles with germinal centers. 

The epithelial cells of mucous membranes play an impor- 
tant role in promoting the immune response by delivering 
small samples of foreign antigen from the lumina of the res- 
piratory, digestive, and urogenital tracts to the underlying 
mucosal-associated lymphoid tissue. This antigen transport 
is carried out by specialized M cells. The structure of the M 
cell is striking: these are flattened epithelial cells lacking the 
microvilli that characterize the rest of the mucous epithe- 
lium. In addition, M cells have a deep invagination, or 
pocket, in the basolateral plasma membrane; this pocket is 
filled with a cluster of B cells, T cells, and macrophages (Fig- 
ure 2-22a). Luminal antigens are endocytosed into vesicles 
that are transported from the luminal membrane to the un- 
derlying pocket membrane. The vesicles then fuse with the 
pocket membrane, delivering the potentially response-acti- 
vating antigens to the clusters of lymphocytes contained 
within the pocket. 

M cells are located in so-called inductive sites — small re- 
gions of a mucous membrane that lie over organized lym- 
phoid follicles (Figure 2-22b). Antigens transported across 
nbrane by M cells can activate B cells within 




tive sites, (a) M cells, located in mucous me 
antigen from the lumen of the digs 
tracts. The antigen is transported ac 
large basolateral pocket, (b) Antiger 
ayer by M cells at an inductive site 



of IgA at indue 
5 membranes, endocytos 
respiratory, and urogenita 



ransported a* 



is the epithelial 
the underlying 



lymphoid follicles. The activ 
ducing plasma cells, which n 
mucosal epithelial layer co 
which many are CD8 + T cell 
ceptor diversity for antigen. 



itains intraepithelial lymphocytes 
that express 78 TCRs with limitec 



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' Immunology 5e: 



these lymphoid follicles. The activated B cells differentiate 
into plasma cells, which leave the follicles and secrete the IgA 
class of antibodies. These antibodies then are transported 
across the epithelial cells and released as secretory IgA into 
the lumen, where they can interact with antigens. 

As described in Chapter 1, mucous membranes are an ef- 
fective barrier to the entrance of most pathogens, which 
thereby contributes to nonspecific immunity. One reason for 
this is that the mucosal epithelial cells are cemented to one 
another by tight junctions that make it difficult for patho- 
gens to penetrate. Interestingly, some enteric pathogens, in- 
cluding both bacteria and viruses, have exploited the M cell 
as an entry route through the mucous-membrane barrier. In 
some cases, the pathogen is internalized by the M cell and 
transported into the pocket. In other cases, the pathogen 
binds to the M cell and disrupts the cell, thus allowing entry 
of the pathogen. Among the pathogens that use M cells in 
these ways are several invasive Salmonella species, Vibrio 
cholerae, and the polio virus. 

Cutaneous-Associated Lymphoid Tissue 

The skin is an important anatomic barrier to the external en- 
vironment, and its large surface area makes this tissue impor- 
tant in nonspecific (innate) defenses. The epidermal (outer) 
layer of the skin is composed largely of specialized epithelial 
cells called keratinocytes. These cells secrete a number of cy- 
tokines that may function to induce a local inflammatory re- 
action. In addition, keratinocytes can be induced to express 
class II MHC molecules and may function as antigen-present- 
ing cells. Scattered among the epithelial-cell matrix of the epi- 
dermis are Langerhans cells, a type of dendritic cell, which 
internalize antigen by phagocytosis or endocytosis. The 
Langerhans cells then migrate from the epidermis to regional 
lymph nodes, where they differentiate into interdigitating 
dendritic cells. These cells express high levels of class II MHC 
molecules and function as potent activators of naive T H cells. 
The epidermis also contains so-called intraepidermal lym- 
phocytes. These are similar to the intraepithelial lymphocytes 
of MALT in that most of them are CD8 + T cells, many of 
which express 78 T-cell receptors, which have limited diver- 
sity for antigen. These intraepidermal T cells are well situated 
to encounter antigens that enter through the skin and some 
immunologists believe that they may play a role in combat- 
ing antigens that enter through the skin. The underlying der- 
mal layer of the skin contains scattered CD4 and CD8 T 
cells and macrophages. Most of these dermal T cells were ei- 
ther previously activated cells or are memory cells. 



Systemic Function of the 
Immune System 

The many different cells, organs, and tissues of the immune 
system are dispersed throughout the body, yet the various 
components communicate and collaborate to produce an ef- 



migrate 11 
lymph cai 
nodes the 
bers of the i 
tiate adapti 



fective response to an infection. An infection that begins in 
one area of the body initiates processes that eventually in- 
volve cells, organs, and tissues distant from the site of 
pathogen invasion. Consider what happens when the skin 
is broken, allowing bacteria to enter the body and initiate 

The tissue damage associated with the injury and infec- 
tion results in an inflammatory response that causes in- 
creased blood flow, vasodilation, and an increase in capillary 
permeability. Chemotactic signals are generated that can 
cause phagocytes and lymphocytes to leave the blood stream 
and enter the affected area. Factors generated during these 
early stages of the infection stimulate the capacity of the 
adaptive immune system to respond. Langerhans cells (den- 
dritic cells found throughout the epithelial layers of the skin 
and the respiratory, gastrointestinal, urinary, and genital 
tracts) can capture antigens from invading pathogens and 
o a nearby lymphatic vessel, where the flow of 
ies them to nearby lymph nodes. In the lymph 
: class II MHC-bearing cells can become mem- 
terdigitating dendritic-cell population and ini- 
immune responses by presenting antigen to T H 
cells. The recognition of antigen by T H cells can have impor- 
tant consequences, including the activation and proliferation 
of T H cells within the node as the T H cells recognize the anti- 
gen, and the secretion by the activated T cells of factors that 
support T-cell-dependent antibody production by B cells 
that may already have been activated by antigen delivered to 
the lymph node by lymph. The antigen-stimulated T H cells 
also release chemotactic factors that cause lymphocytes to 
leave the blood circulation and enter the lymph node 
through the endothelium of the postcapillary venules. Lym- 
phocytes that respond to the antigen are retained in the 
lymph node for 48 hours or so as they undergo activation 
and proliferation before their release via the node's efferent 
lymphatic vessel. 

Once in the lymph, the newly released activated lympho- 
cytes can enter the bloodstream via the subclavian vein. 
Eventually, the circulation carries them to blood vessels near 
the site of the infection, where the inflammatory process 
makes the vascular endothelium of the nearby blood vessels 
more adherent for activated T cells and other leukocytes (see 
Chapter 15). Chemotactic factors that attract lymphocytes, 
macrophages, and neutrophils are also generated during the 
inflammatory process, promoting leukocyte adherence to 
nearby vascular epithelium and leading leukocytes to the site 
of the infection. Later in the course of the response, 
pathogen-specific antibodies produced in the node are also 
carried to the bloodstream. Inflammation aids the delivery of 
the anti-pathogen antibody by promoting increased vascular 
permeability, which increases the flow of antibody-contain- 
ing plasma from the blood circulation to inflamed tissue. The 
result of this network of interactions among diffusible mole- 
cules, cells, organs, the lymphatic system, and the circulatory 
system is an effective and focused immune response to an 



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' Immunology 5e: 



Lymphoid Cells and Organs — 
Evolutionary Comparisons 

While innate systems of immunity are seen in invertebrates 
and even in plants, the evolution of lymphoid cells and or- 
gans evolved only in the phylum Vertebrata. Consequently, 



adaptive immunity, which is mediated by antibodies and T 
cells, is only seen in this phylum. However, as shown in Fig- 
ure 2-23, the kinds of lymphoid tissues seen in different or- 
ders of vertebrates differ. 

As one considers the spectrum from the earliest verte- 
brates, the jawless fishes (Agnatha), to the birds and mam- 
mals, evolution has added organs and tissues with i: 




P*-' Evolutionary distribution of lymphoid tissues. The 
presence and location of lymphoid tissues in several major orders o 
vertebrates are shown. Although they are not shown in the diagram 
cartilaginous fish such as sharks and rays have GALT, thymus, and i 
spleen. Reptiles also have GALT, thymus, and spleen and they alsc 



may have lymph nodes that participate in immunological n 
Whether bone marrow is involved in the generation of lymphocytes 
in reptiles is under investigation. [Adapted from Dupasquier and 
M. Flajnik, 1999. In Fundamental Immunology 4th ed., W. E. Paul, 
ed., Lippincott-Raven, Philadelphia.] 



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' Immunology 5e: 



functions but has tended to retain those evolved by earlier or- 
ders. While all have gut-associated lymphoid tissue (GALT) 
and most have some version of a spleen and thymus, not all 
have blood-cell-forming bone marrow or lymph nodes, and 
the ability to form germinal centers is not shared by all. The 
differences seen at the level of organs and tissues are also re- 
flected at the cellular level. Lymphocytes that express anti- 
gen-specific receptors on their surfaces are necessary to 
mount an adaptive immune response. So far, it has not been 
possible to demonstrate the presence of T or B lymphocytes 
in the jawless fishes, and attempts to demonstrate an adaptive 
immune response in lampreys and hagfish, members of the 
order Agnatha, have failed. In fact, only jawed vertebrates 
(Gnathosomata), of which the cartilaginous fish (sharks, 
rays) are the earliest example, have B and T lymphocytes and 
support adaptive immune responses. 



SUMMARY 

■ The cells that participate in the immune response are white 
blood cells, or leukocytes. The lymphocyte is the only cell 
to possess the immunologic attributes of specificity, diver- 
sity, memory, and self/nonself recognition. 

■ Many of the body's cells, tissues, and organs arise from the 
progeny of different stem-cell populations. The division of 
a stem cell can result in the production of another stem cell 
and a differentiated cell of a specific type or group. 

■ All leukocytes develop from a common multipotent 
hematopoietic stem cell during hematopoiesis. Various 
hematopoietic growth factors (cytokines) induce prolifer- 
ation and differentiation of the different blood cells. The 
differentiation of stem cells into different cell types re- 
quires the expression of different lineage- determining 
genes. A number of transcription factors play important 
roles in this regard. 

■ Hematopoiesis is closely regulated to assure steady-state 
levels of each of the different types of blood cell. Cell divi- 
sion and differentiation of each of the lineages is balanced 
by programmed cell death. 

■ There are three types of lymphocytes: B cells, T cells, and 
natural killer cells (NK cells). NK cells are much less abun- 
dant than B and T cells, and most lack a receptor that is 
specific for a particular antigen. However, a subtype of NK 
cells, NK1-T cells, have both T-cell receptors and many of 
the markers characteristic of NK cells. The three types of 
lymphoid cells are best distinguished on the basis of func- 
tion and the presence of various membrane molecules. 

■ Naive B and T lymphocytes (those that have not encoun- 
tered antigen) are small resting cells in the G phase of the 
cell cycle. After interacting with antigen, these cells enlarge 
into lymphoblasts that proliferate and eventually differen- 
tiate into effector cells and memory cells. 

■ Macrophages and neutrophils are specialized for the 
phagocytosis and degradation of antigens (see Figure 2-9). 



Phagocytosis is facilitated by opsonins such as antibody, 
which increase the attachment of antigen to the membrane 
of the phagocyte. 

■ Activated macrophages secrete various factors that regu- 
late the development of the adaptive immune response 
and mediate inflammation (see Table 2-7). Macrophages 
also process and present antigen bound to class II MHC 
molecules, which can then be recognized by T H cells. 

■ Basophils and mast cells are nonphagocytic cells that re- 
lease a variety of pharmacologically active substances and 
play important roles in allergic reactions. 

■ Dendritic cells capture antigen. With the exception of fol- 
licular dendritic cells, these cells express high levels of class 
II MHC molecules. Along with macrophages and B cells, 
dendritic cells play an important role in T H -cell activation 
by processing and presenting antigen bound to class II 
MHC molecules and by providing the required co-stimula- 
tory signal. Follicular dendritic cells, unlike the others, fa- 
cilitate B-cell activation but play no role in T-cell activation. 

■ The primary lymphoid organs provide sites where lympho- 
cytes mature and become antigenically committed. T lym- 
phocytes mature within the thymus, and B lymphocytes 
arise and mature within the bone marrow of humans, mice, 
and several other animals, but not all vertebrates. 

■ Primary lymphoid organs are also places of selection 
where many lymphocytes that react with self antigens are 
eliminated. Furthermore, the thymus eliminates thymo- 
cytes that would mature into useless T cells because their 
T-cell receptors are unable to recognize self-MHC. 

■ The lymphatic system collects fluid that accumulates in tis- 
sue spaces and returns this fluid to the circulation via the 
left subclavian vein. It also delivers antigens to the lymph 
nodes, which interrupt the course of lymphatic vessels. 

■ Secondary lymphoid organs capture antigens and provide 
sites where lymphocytes become activated by interaction 
with antigens. Activated lymphocytes undergo clonal pro- 
liferation and differentiation into effector cells. 

■ There are several types of secondary lymphoid tissue: 
lymph nodes, spleen, the loose clusters of follicles, and 
Peyer's patches of the intestine, and cutaneous-associated 
lymphoid tissue. Lymph nodes trap antigen from lymph, 
spleen traps blood-borne antigens, intestinal-associated 
lymphoid tissues (as well as other secondary lymphoid tis- 
sues) interact with antigens that enter the body from the 
gastrointestinal tract, and cutaneous-associated lymphoid 
tissue protects epithelial tissues. 

■ An infection that begins in one area of the body eventually 
involves cells, organs, and tissues that may be distant from 
the site of pathogen invasion. Antigen from distant sites 
can arrive at lymph nodes via lymph and dendritic cells, 
thereby assuring activation of T cells and B cells and release 
of these cells and their products to the circulation. Inflam- 
matory processes bring lymphocytes and other leukocytes 
to the site of infection. Thus, although dispersed through- 



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out the body, the components of the h 
municate and collaborate to produce an effective response 
to infection. 
■ Vertebrate orders differ greatly in the kinds of lymphoid 
organs, tissues, and cells they possess. The most primitive 
vertebrates, the jawless fishes, have only gut-associated 
lymphoid tissues, lack B and T cells, and cannot mount 
adaptive immune responses. Jawed vertebrates possess a 
greater variety of lymphoid tissues, have B and T cells, and 
display adaptive immunity. 

References 

Appelbaum, R R. 1996. Hematopoietic stem cell transplanta- 
tion. In Scientific American Medicine. D. Dale and D. Feder- 
man, eds. Scientific American Publishers, New York. 

Banchereau J., R Briere, C. Caux, J. Davoust, S. Lebecque, Y. J. 
Liu, B. Pulendran, and K. Palucka. 2000. Immunobiology of 
dendritic cells. Annu. Rev. Immunology. 18:767. 

Bendelac, A., M. N. Rivera, S-H. Park, and J. H. Roark. 1997. 
Mouse CDl-specific NK1 T cells: Development, specificity 
and function. Annu. Rev. Immunol. 15:535. 

Clevers, H. C, and R. Grosschedl. 1996. Transcriptional control 
of lymphoid development: lessons from gene targeting. Im- 
munol. Today 17:336. 

Cory, S. 1995. Regulation of lymphocyte survival by the BCL-2 
gene family. Annu. Rev. Immunol. 12:513. 

Ganz, T, and R. I. Lehrer. 1998. Antimicrobial peptides of verte- 
brates. Curr. Opin. Immunol. 10:41. 

Liu, Y. J. 2001. Dendritic cell subsets and lineages, and their func- 
tions in innate and adaptive immunity. Cell 106:259. 

Melchers, E, and A. Rolink. 1999. B-lymphocyte development 
and biology. 1 Immunology, 4th ed., W. E. Paul, 

ed., p. 183. Lippincott-Raven, Philadelphia. 

Nathan, C, and M. U. Shiloh. 2000. Reactive oxygen and nitro- 
gen intermediates in the relationship between mammalian 
hosts and microbial pathogens. Proc. Nad. Acad. Sci. 97:8841. 



n cells for medicine. Sci. 



Pedersen, R. A. 1999. Embryonic s 
Am. 280:68. 

Osborne, B. A. 1996. Apoptosis and the maintenance of home- 
ostasis in the immune system. Curr. Opin. Immunol. 8:245. 

Picker, L. J., and M. H. Siegelman. 1999. Lymphoid tissues and 
organs. In Fundamental Immunology, 4thed.,W. E.Paul, ed., p. 
145. Lippincott-Raven, Philadelphia. 

Rothenberg, E. V. 2000. Stepwise specification of lymphocyte de- 
velopmental lineages. Current Opin. Gen. Dev. 10:370. 

Ward, A. C, D. M. Loeb, A. A. Soede-Bobok, I. P. Touw, and A. D. 
Friedman. 2000. Regulation of granulopoiesis by transcription 
factors and cytokine signals. Leukemia 14:973. 

Weissman, I. L. 2000. Translating stem and progenitor cell 
biology to the clinic: barriers and opportunities. Science 
287:1442. 



USEFUL WEB SITES 
http://www.ncbi.nlm. 



nih.gov/pro 



The PROW Guides are authoritative short, structured reviews 
on proteins and protein families that bring together the most 
relevant information on each molecule into a single docu- 
ment of standardized format. 



http://hms.medweb.har\ 
AtlasTOC.htm 

This brilliantly illustrated atlas of normal and abnormal 
blood cells informatively displayed as stained cell smears has 
been assembled to help train medical students at the Harvard 
Medical School to recognize and remember cell morphology 
that is associated with many different pathologies, including 
leukemias, anemias, and even malarial infections. 



ird.edu/nmw/HS_heme/ 



http://www.nih.gov/news/stemcell/primer.htm 

This site provides a brief, but informative introduction to 
stem cells, including their importance and promise as tools 
for research and therapy. 

http://www.nih.gov/news/stemcell/scireport.htm 









e presentatio 

n interesting and well-refer- 



A well written and comprehen 
and their biology is presented ii 
enced monograph. 

Study Questions 

Clinical Focus Question The T and B cells that differentiate 
from hematopoietic stem cells recognize as self the bodies in 
which they differentiate. Suppose a woman donates HSCs to a 
genetically unrelated man whose hematopoietic system was to- 
tally destroyed by a combination of radiation and chemother- 
apy. Suppose further that, although most of the donor HSCs 
differentiate into hematopoietic cells, some differentiate into 
cells of the pancreas, liver, and heart. Decide which of the fol- 
lowing outcomes is likely and justify your choice. 

a. The T cells from the donor HSCs do not attack the pancre- 
atic, heart, and liver cells that arose from donor cells, but 
mount a GVH response against all of the other host cells. 

b. The T cells from the donor HSCs mount a GVH response 
against all of the host cells. 

c. The T cells from the donor HSCs attack the pancreatic, heart, 
and liver cells that arose from donor cells, but fail to mount a 
GVH response against all of the other host cells. 

d. The T cells from the donor HSCs do not attack the pancreatic, 
heart, and liver cells that arose from donor cells and fail to 
mount a GVH response against all of the other host cells. 



1 . Explain why each of the following 



only antigen a: 



a. All T H cells express CD4 and recogniz 
dated with class II MHC molecules. 

b. The pluripotent stem cell is one of the most abundant cell 
types in the bone marrow. 

c. Activation of macrophages increases their expression of 
class I MHC molecules, making the cells present antigen 
more effectively. 



Go to www.whfreeman.com/immunology 
Review and quiz of key terms 



{jA Self-Test 



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' Immunology 5e: 



d. Lymphoid follicles are present only in the spleen and 
lymph nodes. 

e. Infection has no influence on the rate of hematopoiesis. 

f. Follicular dendritic cells can process and present antigen 
to T lymphocytes. 

g. All lymphoid cells ha 
their membrane. 

h. All vertebrates generate 
i. All vertebrates produc 
produce both. 

!. For each of the following situations, indicate which type(s) 
of lymphocyte(s), if any, would be expected to proliferate 
rapidly in lymph nodes and where in the nodes they would 



antigen-specific receptor 



lymphocytes in bone 
5 or T lymphocytes ; 



a. It filters antigens out of the blood. 

b. The marginal zone is rich in T cells, and the periarteriolar 
lymphoid sheath (PALS) is rich in B cells. 

is germinal centers. 

ns to remove old and defective red blood cells. 



d. It functi 

e. Lymphatic vessels draining 
spleen. 

f. Lymph node but not splee 
knockout of the Ikaros gene 






r the 



function is affected by a 



1 4. For each type of cell indicated (a-p), select the most appro- 
priate description (1-16) listed below. Each description may 
be used once, more than once, or not at all. 



. Normal n 



e immunized with a soluble protein a 



b. Normal mouse with a viral infection 

c. Neonatally thymectomized mouse immunized with a 
protein antigen 

d. Neonatally thymectomized mouse immunized with the 
thymus-independent antigen bacterial lipopolysaccha- 
ride (LPS), which does not require the aid of T H cells to 
activate B cells 

3. List the primary lymphoid organs and summarize their 
functions in the immune response. 

4. List the secondary lymphoid organs and summarize their 
functions in the immune response. 

5. What are the two primary characteristics that distinguish 
hematopoietic stem cells and progenitor cells? 



6. What are the tv 

7. What do nude l 



j primary roles of the thymus? 

lice and humans with DiGeorge's syndrome 

8. At what age does the thymus reach its maximal size? 

a. During the first year of life 

b. Teenage years (puberty) 

c. Between 40 and 50 years of age 

d. After 70 years of age 

9. Preparations enriched in hematopoietic stem cells are useful 
for research and clinical practice. In Weissman's method for 
enriching hematopoietic stem cells, why is it necessary to use 
lethally irradiated mice to demonstrate enrichment? 

1 0. What effect does thymectomy have on a neonatal mouse? 
On an adult mouse? Explain why these effects differ. 

1 1 . What effect would removal of the bursa of Fabricius (bur- 
sectomy) have on chickens? 

12. Some microorganisms (e.g., Neisseria gonorrhoeae, My- 
cobacterium tuberculosis, and Candida albicans) are classified 
as intracellular pathogens. Define this term and explain why 
the immune response to these pathogens differs from that to 
other pathogens such as Staphylococcus aureus and Strepto- 
coccus pneumoniae. 

13. Indicate whether each of the following statements about the 
spleen is true or false. If you think a statement is false, ex- 
plain why. 



_ Common myeloid progenitor cells 

_ Monocytes 

_ Eosinophils 

_ Dendritic cells 

_ Natural killer (NK) cells 

_ Kupffer cells 

_ Lymphoid dendritic cell 

_ Mast cells 

_ Neutrophils 

_ M cells 

_ Bone-marrow stromal cells 

_ Lymphocytes 

_NK1-Tcell 

_ Microglial cell 

_ Myeloid dendritic cell 

_ Hematopoietic stem cell 



Descriptions 

( 1 ) Major cell type presenting antigen to T H cells 

(2) Phagocytic cell of the central nervous system 

(3) Phagocytic cells important in the body's defense 
against parasitic organisms 

(4) Macrophages found in the liver 

(5) Give rise to red blood cells 

(6) An antigen-presenting cell derived from monocytes 
that is not phagocytic 

(7) Generally first cells to arrive at site of inflammation 

(8) Secrete colony-stimulating factors (CSFs) 

(9) Give rise to thymocytes 

(10) Circulating blood cells that differentiate into macro- 
phages in the tissues 

(11) An antigen-presenting cell that arises from the same 
precursor as a T cell but not the same as a macrophage 

(12) Cells that are important in sampling antigens of the 
intestinal lumen 

(13) Nonphagocytic granulocytic cells that release various 
pharmacologically active substances 

(14) White blood cells that migrate into the tissues and play 
an important role in the development of a 

(15) These cells sometimes recognize their targets with the 
aid of an antigen-specific cell-surface receptor and 
sometimes by mechanisms that resemble those of nat- 
ural killer cells. 

(16) Members of this category of cells are not found in jaw- 
less fishes. 



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Antigens 



UBSTANCES THAT CAN BE RECOGNIZED BY THE 

immunoglobulin receptor of B cells, or by the T- 
cell receptor when complexed with MHC, are 
called antigens. The molecular properties of antigens and 
the way in which these properties ultimately contribute to 
e activation are central to our understanding of the 
e system. This chapter describes some of the molecu- 
lar features of antigens recognized by B or T cells. The chap- 
ter also explores the contribution made to immunogenicity 
by the biological system of the host; ultimately the biological 
system determines whether a molecule that combines with a 
B or T cell's antigen-binding receptor can then induce an im- 
mune response. Fundamental differences in the way B and T 
lymphocytes recognize antigen determine which molecular 
features of an antigen are recognized by each branch of the 
immune system. These differences are also examined in this 
chapter. 




■ Immunogenicity Versus Antigenicity 

■ Factors That Influence Immunogenicity 

■ Epitopes 

■ Haptens and the Study of Antigenicity 

■ Pattern-Recognition Receptors 



Immunogenicity Versus Antigenicity 

Immunogenicity and antigenicity are related but distinct 
immunologic properties that sometimes are confused. Im- 
munogenicity is the ability to induce a humoral and/or cell- 
mediated immune response: 

B cells + antigen — > effector B cells + memory B cells 

I 
(plasma cells) 

T cells + antigen — > effector T cells + memory T cells 
i 
(e.g.,CTLs,T H s) 

Although a substance that induces a specific immune re- 
sponse is usually called an antigen, it is more appropriately 
called an immunogen. 

Antigenicity is the ability to combine specifically with 
the final products of the above responses (i.e., antibodies 
and/or cell-surface receptors). Although all molecules that 
have the property of immunogenicity also have the property 
of antigenicity, the reverse is not true. Some small molecules, 
called haptens, are antigenic but incapable, by themselves, of 
inducing a specific immune response. In other words, they 
lack immunogenicity. 



Factors That Influence 
Immunogenicity 

To protect against infectious disease, the immune system 
must be able to recognize bacteria, bacterial products, fungi, 
parasites, and viruses as immunogens. In fact, the immune 
system actually recognizes particular macromolecules of an 
infectious agent, generally either proteins or polysaccharides. 
Proteins are the most potent immunogens, with polysaccha- 
rides ranking second. In contrast, lipids and nucleic acids of 
an infectious agent generally do not serve as immunogens 
unless they are complexed with proteins or polysaccharides. 
Immunologists tend to use proteins or polysaccharides as 
immunogens in most experimental studies of humoral im- 
munity (Table 3-1). For cell-mediated immunity, only pro- 
teins and some lipids and glycolipids serve as immunogens. 
These molecules are not recognized directly. Proteins must 
first be processed into small peptides and then presented to- 
gether with MHC molecules on the membrane of a cell be- 
fore they can be recognized as immunogens. Recent work 
shows that those lipids and glycolipids that can elicit cell- 
mediated immunity must also be combined with MHC-like 
membrane molecules called CD1 (see Chapter 8). 



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™ LE ,,[ 


Antigen 


Approximate molecular m 


ss (Da) 


(BCC) 


150,000 




Bovine serum albumin 
(BSA) 


69,000 




Flagellin (monomer) 


40,000 




Hen egg-white lysozyme 
(HEL) 


15,000 




Keyhole limpet hemocyanin 
(KLH) 


>2,000,000 




Ovalbumin (OVA) 


44,000 




Sperm whale myoglobin 
(SWM) 


17,000 




Tetanus toxoid (TT) 


150,000 





into a cow but is strongly immunogenic when injected into a 
rabbit. Moreover, BSA would be expected to exhibit greater 
immunogenicity in a chicken than in a goat, which is more 
closely related to bovines. There are some exceptions to this 
rule. Some macromolecules (e.g., collagen and cytochrome 
c) have been highly conserved throughout evolution and 
therefore display very little immunogenicity across diverse 
species lines. Conversely, some self-components (e.g., 
corneal tissue and sperm) are effectively sequestered from 
the immune system, so that if these tissues are injected even 
into the animal from which they originated, they will func- 
tion as immunogens. 

MOLECULAR SIZE 

There is a correlation between the size of a macromolecule 
and its immunogenicity. The most active immunogens tend 
to have a molecular mass of 100,000 daltons (Da). Generally, 
substances with a molecular mass less than 5000-10,000 Da 
are poor immunogens, although a few substances with a 
molecular mass less than 1000 Da have proven to be im- 
munogenic. 



Immunogenicity is not an intrinsic property of an antigen 
but rather depends on a number of properties of the particu- 
lar biological system that the antigen encounters. The next 
two sections describe the properties that most immunogens 
share and the contribution that the biological system makes 
to the expression of immunogenicity. 

The Nature of the Immunogen 
Contributes to Immunogenicity 

Immunogenicity is determined, in part, by four properties of 
the immunogen: its foreignness, molecular size, chemical 
composition and complexity, and ability to be processed and 
presented with an MHC molecule on the surface of an anti- 
gen-presenting cell or altered self-cell. 

FOREIGNNESS 

In order to elicit an immune response, a molecule must be 
recognized as nonself by the biological system. The capacity 
to recognize nonself is accompanied by tolerance of self, a 
specific unresponsiveness to self antigens. Much of the ability 
to tolerate self antigens arises during lymphocyte develop- 
ment, during which immature lymphocytes are exposed to 
self-components. Antigens that have not been exposed to im- 
mature lymphocytes during this critical period may be later 
recognized as nonself, or foreign, by the immune system. 
When an antigen is introduced into an organism, the degree 
of its immunogenicity depends on the degree of its foreign- 
ness. Generally, the greater the phylogenetic distance be- 
tween two species, the greater the structural (and therefore 
the antigenic) disparity between them. 

For example, the common experimental antigen bovine 
serum albumin (BSA) is not immunogenic when injected 



CHEMICAL COMPOSITION AND HETEROGENEITY 
Size and foreignness are not, by themselves, sufficient to 
make a molecule immunogenic; other properties are needed 
as well. For example, synthetic homopolymers (polymers 
composed of a single amino acid or sugar) tend to lack im- 
munogenicity regardless of their size. Studies have shown 
that copolymers composed of different amino acids or sugars 
are usually more immunogenic than homopolymers of their 
constituents. These studies show that chemical complexity 
contributes to immunogenicity. In this regard it is notable 
that all four levels of protein organization — primary, sec- 
ondary, tertiary, and quaternary — contribute to the struc- 
tural complexity of a protein and hence affect its immuno- 
genicity (Figure 3-1). 

LIPIDS AS ANTIGENS 

Appropriately presented lipoidal antigens can induce B- and 
T-cell responses. For the stimulation of B-cell responses, 
lipids are used as haptens and attached to suitable carrier 
molecules such as the proteins keyhole limpet hemocyanin 
(KLH) or bovine serum albumin (BSA) . By immunizing with 
these lipid-protein conjugates it is possible to obtain anti- 
bodies that are highly specific for the target lipids. Using this 
approach, antibodies have been raised against a wide variety 
of lipid molecules including steroids, complex fatty-acid de- 
rivatives, and fat-soluble vitamins such as vitamin E. Such 
antibodies are of considerable practical importance since 
many clinical assays for the presence and amounts of med- 
ically important lipids are antibody-based. For example, a 
determination of the levels of a complex group of lipids 
known as leukotrienes can be useful in evaluating asthma pa- 
tients. Prednisone, an immunosuppressive steroid, is often 
given as part of the effort to prevent the rejection of a trans- 



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- Lys - Ala - His - Gly - Lys - Lys - Val - Leu 
Amino acid sequence 
leptide chain 



PRIMARY STRUCTURE 




a helix ^r |3 pleated sheet 

SECONDARY STRUCTURE 





Monomeric polypeptide molecule 
TERTIARY STRUCTURE 



k The four levels of protein organizational 
The linear arrangement of amino acids constitutes the primary s 
ture. Folding of parts of a polypeptide chain into regular struct 
(e.g., a helices and f3 pleated sheets) generates the secondary s 
ture. Tertiary structure refers to the folding of regions between 



Dimeric protein molecule 
QUATERNARY STRUCTURE 

jndary features to give the overall shape of the molecule or par 
t (domains) with specific functional properties. Quaternary s 
:ure results from the association of two or more polypeptide d 
nto a single polymeric protein molecule. 



planted organ. The achievement and maintenance of ade- 
quate blood levels of this and other immunosuppressive 
drugs is important to a successful outcome of transplanta- 
tion, and antibody-based immunoassays are routinely used 
to make these evaluations. The extraordinary sensitivity and 
specificity of assays based on the use of anti-lipid antibodies 
is illustrated by Table 3-2, which shows the specificity of an 
antibody raised against leukotriene C 4 . This antibody allows 
the detection of as little as 16-32 picograms per ml of 
leukotriene C 4 . Because it has little or no reactivity with sim- 
ilar compounds, such as leukotriene D 4 or leukotriene E 4 , it 
can be used to assay leukotriene C 4 in samples that contain 
this compound and a variety of other structurally related 
lipids. 

T cells recognize peptides derived from protein antigens 
when they are presented as peptide-MHC complexes. How- 
ever, some lipids can also be recognized by T cells. Lipoidal 



compounds such as glycolipids and some phospholipids can 
be recognized by T-cell receptors when presented as com- 
plexes with molecules that are very much like MHC mole- 
cules. These lipid-presenting molecules are members of the 
CD1 family (see Chapter 8) and are close structural relatives 
of class I MHC molecules. The lipid molecules recognized by 
the CD 1 -T-cell receptor system all appear to share the com- 
mon feature of a hydrophobic portion and a hydrophilic head 
group. The hydrophobic portion is a long-chain fatty acid or 
alcohol and the hydrophilic head group is composed of highly 
polar groups that often contain carbohydrates. Recognition of 
lipids is a part of the immune response to some pathogens, 
and T cells that recognize lipids arising from Mycobacterium 
tuberculosis and Mycobacterium leprae, which respectively 
cause tuberculosis and leprosy, have been isolated from hu- 
mans infected by these mycobacteria. More about the presen- 
tation of lipoidal antigens can be found in Chapter 8. 



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Antibody reactivity* 
(on scale of 1 to 100) 



Leukotriene C 4 



Leukotriene D 4 



Prostaglandin D 2 




"The reactivity of the antibody with th 






SUSCEPTIBILITY TO ANTIGEN PROCESSING 
AND PRESENTATION 

The development of both humoral and cell-mediated im- 
mune responses requires interaction of T cells with antigen 
that has been processed and presented together with MHC 
molecules. Large, insoluble macromolecules generally are 
more immunogenic than small, soluble ones because the 
larger molecules are more readily phagocytosed and 
processed. Macromolecules that cannot be degraded and 
presented with MHC molecules are poor immunogens. This 
can be illustrated with polymers of D-amino acids, which are 
stereoisomers of the naturally occurring L-amii 
cause the degradative enzymes within antigei 
cells can degrade only proteins containing l-e 
polymers of D-amino acids cannot be processed 



o acids. Be- 
-presenting 

nd thus are 



The Biological System Contributes 
to Immunogenicity 

Even if a macromolecule has the properties that contribute to 
immunogenicity, its ability to induce an immune response 
will depend on certain properties of the biological system 
that the antigen encounters. These properties include the 
genotype of the recipient, the dose and route of antigen ad- 
ministration, and the administration of substances, called 
adjuvants, that increase immune responses. 

GENOTYPE OFTHE RECIPIENT ANIMAL 
The genetic constitution (genotype) of an immunized ani- 
mal influences the type of immune response the animal 
manifests, as well as the degree of the response. For example, 
Hugh McDevitt showed that two different inbred strains of 



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mice responded very differently to a synthetic polypeptide 
immunogen. After exposure to the immunogen, one strain 
produced high levels of serum antibody, whereas the other 
strain produced low levels. When the two strains were 
crossed, the F[ generation showed an intermediate response 
to the immunogen. By backcross analysis, the gene control- 
ling immune responsiveness was mapped to a subregion of 
the major histocompatibility complex (MHC). Numerous 
experiments with simple defined immunogens have demon- 
strated genetic control of immune responsiveness, largely 
confined to genes within the MHC. These data indicate that 
MHC gene products, which function to present processed 
antigen to T cells, play a central role in determining the de- 
gree to which an animal responds to an immunogen. 

The response of an animal to an antigen is also influenced 
by the genes that encode B-cell and T-cell receptors and by 
genes that encode various proteins involved in immune reg- 
ulatory mechanisms. Genetic variability in all of these genes 
affects the immunogenicity of a given macromolecule in dif- 
ferent animals. These genetic contributions to immuno- 
genicity will be described more fully in later chapters. 

IMMUNOGEN DOSAGE AND ROUTE OF ADMINISTRATION 
Each experimental immunogen exhibits a particular dose-re- 
sponse curve, which is determined by measuring the 
mune response to different doses and different adminis- 
tration routes. An antibody response is measured by deter- 
mining the level of antibody present in the serum of immu- 
nized animals. Evaluating T-cell responses is less simple but 
may be determined by evaluating the increase in the number 
of T cells bearing TCRs that recognize the immunogen. Some 
combination of optimal dosage and route of adj 
will induce a peak immune response in a given a 

An insufficient dose will not stimulate an 
sponse either because it fails to activate enough lymphocytes 
or because, in some cases, certain ranges of low doses can in- 
duce a state of immunologic unresponsiveness, or tolerance. 
The phenomenon of tolerance is discussed in chapters 10 
and 21. Conversely, an excessively high dose can also induce 
tolerance. The immune response of mice to the purified 
pneumococcal capsular polysaccharide illustrates the impor- 
tance of dose. A 0.5 mg dose of antigen fails to induce an im- 
mune response in mice, whereas a thousand-fold lower dose 
of the same antigen (5 X 10~ 4 mg) induces a humoral anti- 
body response. A single dose of most experimental immuno- 
gens will not induce a strong response; rather, repeated 
administration over a period of weeks is usually required. 
Such repeated administrations, or boosters, increase the 
clonal proliferation of antigen-specific T cells or B cells and 
thus increase the lymphocyte populations specific for the im- 
munogen. 

Experimental immunogens are generally administered 
parenterally (para, around; enteric, gut) — that is, by routes 
other than the digestive tract. The following 



Intravenous (iv): into a vein 
Intradermal (id): into the skin 
Subcutaneous (sc): beneath the skin 
Intramuscular (im): into a muscle 
Intraperitoneal (ip): into the peritoneal c 






The administration route strongly influences which immune 
organs and cell populations will be involved in the response. 
Antigen administered intravenously is carried first to the 
spleen, whereas antigen administered subcutaneously moves 
first to local lymph nodes. Differences in the lymphoid cells 
that populate these organs may be reflected in the subsequent 
immune response. 

ADJUVANTS 

Adjuvants (from Latin adjuvare, to help) are substances that, 
when mixed with an antigen and injected with it, enhance the 
immunogenicity of that antigen. Adjuvants are often used to 
boost the immune response when an antigen has low im- 
munogenicity or when only small amounts of an antigen are 
available. For example, the antibody response of mice to im- 
munization with BSA can be increased fivefold or more if the 
BSA is administered with an adjuvant. Precisely how adju- 
vants augment the immune response is not entirely known, 
but they appear to exert one or more of the following effects 
(Table 3-3): 

■ Antigen persistence is prolonged. 

■ Co-stimulatory signals are enhanced. 

■ Local inflammation is increased. 

■ The nonspecific proliferation of lymphocytes is 
stimulated. 

Aluminum potassium sulfate (alum) prolongs the persis- 
tence of antigen. When an antigen is mixed with alum, the 
salt precipitates the antigen. Injection of this alum precipitate 
results in a slower release of antigen from the injection site, so 
that the effective time of exposure to the antigen increases 
from a few days without adjuvant to several weeks with the 
adjuvant. The alum precipitate also increases the size of the 
antigen, thus increasing the likelihood of phagocytosis. 

Water-in-oil adjuvants also prolong the persistence of 
antigen. A preparation known as Freund's incomplete ad- 
juvant contains antigen in aqueous solution, mineral oil, 
and an emulsifying agent such as mannide monooleate, 
which disperses the oil into small droplets surrounding the 
antigen; the antigen is then released very slowly from the 
site of injection. This preparation is based on Freund's 
complete adjuvant, the first deliberately formulated 
highly effective adjuvant, developed by Jules Freund many 
years ago and containing heat-killed Mycobacteria as an 
additional ingredient. Muramyl dipeptide, a component of 
the mycobacterial cell wall, activates macrophages, making 



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POSTULATED MODE OF ACTIO 



Adjuvant 



Prolongs 


Enhances 


Induces 


Stimulates 


antigen 


co-stimulatory 


granuloma 


lymphocytes 


persistence 


signal 


formation 


nonspecifically 



Freund's incomplete adjuvant 

Freund's complete adjuvant 

Aluminum potassium sulfate (alum) 

Mycobacterium tuberculosis 

Bordetella pertussis 

Bacterial lipopolysaccharide (LPS) 

Synthetic polynucleotides (poly IC/poly AU) 



Freund's complete adjuvant far more potent than the in- 
complete form. Activated macrophages are more phago- 
cytic than unactivated macrophages and express higher 
levels of class II MHC molecules and the membrane mole- 
cules of the B7 family. The increased expression of class II 
MHC increases the ability of the antigen-presenting cell to 
present antigen to T H cells. B7 molecules on the antigen- 
presenting cell bind to CD28, a cell-surface protein on T H 
cells, triggering co-stimulation, an enhancement of the T- 
cell immune response. Thus, antigen presentation and the 
requisite co-stimulatory signal usually are increased in the 
presence of adjuvant. 

Alum and Freund's adjuvants also stimulate a local, 
chronic inflammatory response that attracts both phagocytes 
and lymphocytes. This infiltration of cells at the site of the 
adjuvant injection often results in formation of a dense, 
macrophage-rich mass of cells called a granuloma. Because 
the macrophages in a granuloma are activated, this mecha- 
nism also enhances the activation of T H cells. 

Other adjuvants (e.g., synthetic polyribonucleotides and 
bacterial lipopolysaccharides) stimulate the nonspecific pro- 
liferation of lymphocytes and thus increase the likelihood of 
antigen-induced clonal selection of lymphocytes. 



Epitopes 



As mentioned in Chapter 1, immune cells do not i 
with, or recognize, an entire immunogen molecule; instead, 
lymphocytes recognize discrete sites on the macromolecule 
called epitopes, or antigenic determinants. Epitopes are the 
immunologically active regions of an immunogen that bind 
to antigen-specific membrane receptors on lymphocytes or 
to secreted antibodies. Studies with small antigens have re- 
vealed that B and T cells recognize different epitopes on the 
same antigenic molecule. For example, when mice were im- 
munized with glucagon, a small human hormone of 29 
amino acids, antibody was elicited to epitopes in the amino- 



terminal portion, whereas the T cells responded only to epi- 
topes in the carboxyl-terminal portion. 

Lymphocytes may interact with a complex antigen on sev- 
eral levels of antigen structure. An epitope on a protein anti- 
gen may involve elements of the primary, secondary, tertiary, 
and even quaternary structure of the protein (see Figure 3-1). 
In polysaccharides, branched chains are commonly present, 
and multiple branches may contribute to the conformation 
of epitopes. 

The recognition of antigens by T cells 
mentally different (Table 3-4). B cells 
gen when it bii 
Because B cells bi 



Ld B cells is funda- 
;nize soluble anti- 
their membrane-bound antibody, 
that is free in solution, the epi- 



topes they recognize tend to be highly accessible si 
exposed surface of the immunogen. As noted previously, 
most T cells recognize only peptides combined with MHC 
molecules on the surface of antigen-presenting cells and al- 
tered self-cells; T-cell epitopes, as a rule, cannot be consid- 
ered apart from their associated MHC molecules. 

Properties of B-Cell Epitopes Are Determined 
by the Nature of the Antigen-Binding Site 

Several generalizations have emerged from studies in which 
the molecular features of the epitope recognized by B cells 
have been established. 

The ability to function as a B-cell epitope is determined by 
the nature of the antigen-binding site on the antibody molecules 
displayed by B cells. Antibody binds to an epitope by weak 
noncovalent interactions, which operate only over short dis- 
tances. For a strong bond, the antibody's binding site and the 
epitope must have complementary shapes that place the in- 
teracting groups near each other. This requirement poses 
some restriction on the properties of the epitope. The size of 
the epitope recognized by a B cell can be no larger than the 
size of the antibody's binding site. For any given antigen-an- 
tibody reaction, the shape of the epitope that can be recog- 
nized by the antibody is determined by the shape assumed by 



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Characteristic 



Interaction with antigen 


Involves binary complex of membrane 
Igand Ag 


Involves ternary complex of T-cell recepto 
and MHC molecule 


r.Ag, 


Binding of soluble antigen 


Yes 


No 




Involvement of MHC molecules 


None required 


Required to display processed antigen 




Chemical nature of antigens 


Protein, polysaccharide, lipid 


Mostly proteins, but some lipids and 
glycolipids presented on MHC-like 




Epitope properties 


Accessible, hydrophilic, mobile peptides 
containing sequential or nonsequential 


Internal linear peptides produced by 
processing of antigen and bound to 
MHC molecules 





the sequences of amino acids in the binding site and the 
chemical environment that they produce. 

Smaller ligands such as carbohydrates, small oligonu- 
cleotides, peptides, and haptens often bind within a deep 
pocket of an antibody. For example, angiotensin II, a small 
octapeptide hormone, binds within a deep and narrow 
groove (725 A 2 ) of a monoclonal antibody specific for the 
hormone (Figure 3-2). Within this groove, the bound pep- 
tide hormone folds into a compact structure with two turns, 
which brings its amino (N-terminal) and carboxyl (C-termi- 
nal) termini close together. All eight amino acid residues of 
the octapeptide are in van der Waals contact with 14 residues 
of the antibody's groove. 

A quite different picture of epitope structure emerges 
from x-ray crystallographic analyses of monoclonal antibod- 
ies bound to globular protein antigens such as hen egg-white 
lysozyme (HEL) and neuraminidase (an envelope glycopro- 
tein of influenza virus). These antibodies m 
the antigen across a large flat face (Figure 3-3). The i 
ing face between antibody and epitope is a flat or undulating 
surface in which protrusions on the epitope or antibody are 
matched by corresponding depressions on the antibody or 
epitope. These studies have revealed that 15-22 amino acids 
on the surface of the antigen make contact with a similar 
number of residues in the antibody's binding site; the surface 
area of this large complementary interface is between 650 A 2 
and 900 A 2 . For these globular protein antigens, then, the 
shape of the epitope is entirely determined by the tertiary 
conformation of the native protein. 

Thus, globular protein antigens and small peptide anti- 
gens interact with antibody in different ways (Figure 3-4). 
Typically, larger areas of protein antigens are engaged by the 
antibody binding site. In contrast, a small peptide such as an- 
giotensin II can fold into a compact structure that occupies 
less space and fits into a pocket or cleft of the binding site. 
This pattern is not unique to small peptides; it extends to the 
binding of low-molecular- weight antigens of various chemi- 
cal types. However, these differences between the binding of 
small and large antigenic determinants do not reflect funda- 
mental differences in the regions of the antibody molecule 



that make up the binding site. Despite differences in the 
binding patterns of small haptens and large antigens, Chap- 
ter 4 will show that all antibody binding sites are assembled 
from the same regions of the antibody molecule — namely, 
parts of the variable regions of its polypeptide chains. 




| Three-dimensional structure of an octapeptide hor- 
mone (angiotensin II) complexed with a monoclonal antibody Fab 
fragment, the antigen-binding unit of the antibody molecule. The an- 
giotensin II peptide is shown in red, the heavy chain in blue, and the 
light chain in purple. [From K. C. Garcia et al., 1992, Science 257:502.] 



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Generation of B-Cell and T-Cell Respor 




| (a) Model of i 
lysozyme (HEL) and Fab fragment of anti-HEL antibody based on x- 
ray diffraction analysis. HEL is shown in green, the Fab heavy chain in 
blue, and the Fab light chain in yellow. A glutamine residue of 
lysozyme (red) fits into a pocket in the Fab fragment, (b) Representa- 
tion of HEL and the Fab fragment when pulled apart showing com- 
plementary surface features, (c) View of the interacting surfaces of 
the Fab fragment and HEL obtained by rotating each of the mole- 
cules. The contacting residues are numbered and shown in red with 
the protruding glutamine (#14) in HEL now shown in white. [From 
A. C. Am it et al., 1986, Science 233:747.] 

The B-cell epitopes on native proteins generally are com- 
posed ofhydrophilic amino acids on the protein surface that are 
topographically accessible to membrane-bound or free anti- 
body. A B-cell epitope must be accessible in order to be able to 
bind to an antibody; in general, protruding regions on the 
surface of the protein are the most likely to be recognized as 
epitopes, and these regions are usually composed of predom- 
inantly hydrophilic amino acids. Amino acid sequences that 



ind antigen shows n 
md depressions (Figure 
q the antigen o 
ds as well as by i< 



are hidden within the interior of a protein often consist of 
predominantly hydrophobic amino acids, and cannot func- 
tion as B-cell epitopes unless the protein is first denatured. In 
the crystallized antigen-antibody complexes analyzed to 
date, the interface between antibody a 
merous complementary protrusions ai 
3-5). Between 15 and 22 amino ac 
the antibody by 75-120 hydroger 
and hydrophobic interactions. 

B-cell epitopes can contain sequential or nonsequential 
amino acids. Epitopes may be composed of sequential con- 
tiguous residues along the polypeptide chain or nonsequen- 
tial residues from segments of the chain brought together by 
the folded conformation of an antigen. Most antibodies 
elicited by globular proteins bind to the protein only when it 
is in its native conformation. Because denaturation of such 
antigens usually changes the structure of their epitopes, anti- 
bodies to the native protein do not bind to the denatured 

Five distinct sequential epitopes, each containing six to 
eight contiguous amino acids, have been found in sperm 
whale myoglobin. Each of these epitopes is on the surface of 
the molecule at bends between the ct-helical regions (Figure 
3 -6a). Sperm whale myoglobin also contains several nonse- 
quential epitopes, or conformational determinants. The 
residues that constitute these epitopes are far apart in the pri- 
mary amino acid sequence but close together in the tertiary 
structure of the molecule. Such epitopes depend on the 



£& jrtfe Afii 

WP^^k *P?^^4 ^Ff^^T 

HyHel-5 HyHel-10 Dl/3 

M&L *^ jfefr 

WvSQ WB^J WS^> 



iff* Models of the variable domains of six Fab fragments 
with their antigen-binding regions shown in purple. The top three an- 
tibodies are specific for lysozyme, a large globular protein. The lower 
three antibodies are specific for smaller molecules or very small seg- 
ments of macromolecules: McPC603 for phosphocholine; BV04for a 
small segment of a single-stranded DNA molecule; and 17/9 for a 
peptide from hemagglutinin, an envelope protein of influenza virus. 
In general, the binding sites for small molecules are deep pockets, 
whereas binding sites for large proteins are flatter, more undulating 
surfaces. [From I. A. Wilson and R. L Stanfield, 1993, Curr. Opin. 
Struc. Biol. 3:773.] 



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:46:385_REB>H 








Compute 


simulat 


on of 


n interaction b 




body 


nd in 


uenza virus 


antigen, 


iglobi 


lar protei 


■ (a) 


The a 


(yello 


/) is sf 


own interac 


ng with the ant 


bodymol 


cule 


theva 


regior 


of the 


heavy chair 


is red, a 


nd the 


variable r 


egio 


ofth 



chain is blue, (b) The complementarity of the two molecules is re- 
vealed by separating the antigen from the antibody by 8 A. [Based on 
x-ray crystallography data collected by P. M. Colman and W. R. Tulip. 
From C.J. V. H. Nossal, 1993, Sci. Am. 269(3):22] 



VISUALIZING CONCEPTS 







Protein antigens usually contain both sequer 
and nonsequential B-cell epitopes, (a) Diagram of sperm whale 
myoglobin showing locations of five sequential B-cell epitopes 
(blue), (b) Ribbon diagram of hen egg-white lysozyme showing 
residues that compose one nonsequential (conformational) epi- 
tope. Residues that contact antibody light chains, heavy chains, or 



shown in red, blue, and white, respectively. These 
residues are widely spaced in the amino acid sequence but are 
brought into proximity by folding of the protein. [Part (a) adapted 
from M. Z. Atassi and A. L Kazim. 1978, Adv. Exp. Med. Biol. 98:9, 
part (b)from W. C. Laveretal., 1990, Cell 61:554.] 



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Generation of B-Cell and T-Cell Respor 



native protein conformation for their topographical struc- 
ture. One well-characterized nonsequential epitope in hen 
egg-white lysozyme (HEL) is shown in Figure 3-6b. Although 
the amino acid residues that compose this epitope of HEL are 
far apart in the primary amino acid sequence, they are 
brought together by the tertiary folding of the protein. 

Sequential and nonsequential epitopes generally behave 
differently when a protein is denatured, fragmented, or re- 
duced. For example, appropriate fragmentation of sperm 
whale myoglobin can yield five fragments, each retaining one 
sequential epitope, as demonstrated by the observation that 
antibody can bind to each fragment. On the other hand, frag- 
mentation of a protein or reduction of its disulfide bonds of- 
ten destroys nonsequential epitopes. For example, HEL has 
four intrachain disulfide bonds, which determine the final 
protein conformation (Figure 3-7a). Many antibodies to 
HEL recognize several epitopes, and each of eight different 
epitopes have been recognized by a distinct antibody. Most of 



these epitopes are conformational determinants dependent 
on the overall structure of the protein. If the intrachain disul- 
fide bonds of HEL are reduced with mercaptoethanol, the 
nonsequential epitopes are lost; for this reason, antibody to 
native HEL does not bind to reduced HEL. 

The inhibition experiment shown in Figure 3-7 nicely 
demonstrates this point. An antibody to a conformational 
determinant, in this example a peptide loop present in native 
HEL, was able to bind the epitope only if the disulfide bond 
that maintains the structure of the loop was intact. Infor- 
mation about the structural requirements of the antibody 
combining site was obtained by examining the ability of 
structural relatives of the natural antigen to bind to that an- 
tibody. If a structural relative has the critical epitopes present 
in the natural antigen, it will bind to the antibody combining 
site, thereby blocking its occupation by the natural antigen. 
In this inhibition assay, the ability of the closed loop to in- 
hibit binding showed that the closed loop was sufficiently 



(a) Hen egg- 




(b) Synthetic loop peptides 



Disulfide bond 




(c) Inhibition of 

loop and anti-loop 



between HEL 



al demonstration that binding of antibody 
to conformational determinants in hen egg-white lysozyme (HEL) 
depends on maintenance of the tertiary structure of the epitopes by 
intrachain disulfide bonds, (a) Diagram of HEL primary structure, in 
which circles represent amino acid residues. The loop (blue circles) 
formed by the disulfide bond between the cysteine residues at posi- 
tions 64 and 80 constitutes one of the conformational determinants 
in HEL. (b) Synthetic open-loop and closed-loop peptides corre- 
sponding to the HEL loop epitope, (c) Inhibition of binding between 
HEL loop epitope and anti-loop antiserum. Anti-loop antiserum was 
first incubated with the natural loop sequence, the synthetic closed- 
loop peptide, or the synthetic open-loop peptide; the ability of the an- 
tiserum to bind the natural loop sequence then was measured. The 
absence of any inhibition by the open-loop peptide indicates that it 
does not bind to the anti-loop antiserum. [Adapted from D. Benjamin 
et al., 1984, Annu. Rev. Immunol. 2:67.] 



A 



A Natural loop 

Closed synthetic loop 
Open synthetic loop 



Ratio of loop inhibitor to anti-loop 



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8536d_ch03_057-075 8/6/02 



' Immunology 5e: 



similar to HEL to be recognized by antibody to native HEL. 
Even though the open loop had the same sequence of amino 
acids as the closed loop, it lacked the epitopes recognized by 
the antibody and therefore was unable to block binding of 
HEL. 

B-cell epitopes tend to be located inflexible regions of an im- 
munogen and ! >ility. John A. Tainer and his col- 

leagues analyzed the epitopes on a number of protein 
antigens (myohemerytherin, insulin, cytochrome c, myoglo- 
bin, and hemoglobin) by comparing the positions of the 
known B-cell epitopes with the mobility of the same 
residues. Their analysis revealed that the major antigenic de- 
terminants in these proteins generally were located in the 
most mobile regions. These investigators proposed that site 
mobility of epitopes maximizes complementarity with the 
antibody's binding site, permitting an antibody to bind with 
an epitope that it might bind ineffectively if it were rigid. 
However, because of the loss of entropy due to binding to a 
flexible site, the binding of antibody to a flexible epitope is 
generally of lower affinity than the binding of antibody to a 
rigid epitope. 

Complex proteins contain multiple overlapping B-cell epi- 
topes, some of which are immunodominant. For many years, it 
was dogma in immunology that each globular protein had a 
small number of epitopes, each confined to a highly accessi- 
ble region and determined by the overall conformation of the 
protein. However, it has been shown more recently that most 
of the surface of a globular protein is potentially antigenic. 
This has been demonstrated by comparing the antigen-bind- 
ing profiles of different monoclonal antibodies to various 
globular proteins. For example, when 64 different mono- 
clonal antibodies to BSA were compared for their ability to 
bind to a panel of 10 different mammalian albumins, 25 dif- 
ferent overlapping antigen-binding profiles emerged, sug- 
gesting that these 64 different antibodies recognized a 
minimum of 25 different epitopes on BSA. Similar findings 
have emerged for other globular proteins, such as myoglobin 
and HEL. 

The surface of a protein, then, presents a large number of 
potential antigenic sites. The subset of antigenic sites on a 
given protein that is recognized by the immune system of an 
animal is much smaller than the potential antigenic reper- 
toire, and it varies from species to species and even among in- 



dividual members of a given species. Within an animal, cer- 
tain epitopes of an antigen are recognized as immunogenic, 
but others are not. Furthermore, some epitopes, called im- 
munodominant, induce a more pronounced immune re- 
sponse than other epitopes in a particular animal. It is highly 
likely that the intrinsic topographical properties of the epi- 
tope as well as the animal's regulatory mechanisms influence 
: of epitopes. 



Antigen-Derived Peptides Are the Key 
Elements of T-Cell Epitopes 

Studies by P. G. H. Gell and Baruj Benacerraf in 1959 sug- 
gested that there was a qualitative difference between the T- 
cell and the B-cell response to protein antigens. Gell and 
Benacerraf compared the humoral (B-cell) and cell-medi- 
ated (T-cell) responses to a series of native and denatured 
protein antigens (Table 3-5). They found that when primary 
immunization was with a native protein, only native protein, 
not denatured protein, could elicit a secondary antibody (hu- 
moral) response. In contrast, both native and denatured pro- 
tein could elicit a secondary cell-mediated response. The 
finding that a secondary response mediated by T cells was in- 
duced by denatured protein, even when the primary immu- 
nization had been with native protein, initially puzzled 
immunologists. In the 1980s, however, it became clear that T 
cells do not recognize soluble native antigen but rather rec- 
ognize antigen that has been processed into antigenic pep- 
tides, which are presented in combination with MHC 
molecules. For this reason, destruction of the conformation 
of a protein by denaturation does not affect its T-cell epi- 

Because the T-cell receptor does not bind free peptides, 
experimental systems for studying T-cell epitopes must in- 
clude antigen-presenting cells or target cells that can display 
the peptides bound to an MHC molecule. 

Antigenic peptides recognized by T cells form trimolecular 
complexes with a T-cell receptor and an MHC molecule (Figure 
3-8). The structures of TCR-peptide-MHC trimolecular 
complexes have been determined by x-ray crystallography 
and are described in Chapter 9. These structural studies of 
class I or class II MHC molecules crystallized with known T- 
cell antigenic peptides has shown that the peptide binds to a 



Secondary immunizatior 



SECONDARY IMMUNE RESPONSE 



Antibody production 



Cell-mediated T DTH response* 



Native protein 
Native protein 



Native protein 
Denatured protein 



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Generation of B-Cell and T-Cell Respor 







i of the t( 



mplex formed 



| Schematic diagra 
between a T-cell receptor (TCR) on a T H cell, an antigen, and a class 
II MHC molecule. Antigens that are recognized by T cells yield pep- 
tides that interact with MHC molecules to form a peptide-M HC com- 
plex that is recognized by the T-cell receptor. As described in later 
chapters, the coreceptor, CD4, on T H cells also interacts with MHC 
molecules. T c cells form similar ternary complexes with class I MHC 
molecules on target cells however, these cells bear MHC-interacting 
CD8 coreceptors. 

cleft in the MHC molecule (see Figure 7-8). Unlike B-cell 
epitopes, which can be viewed strictly in terms of their ability 
to interact with antibody, T-cell epitopes must be viewed in 
terms of their ability to interact with both a T-cell receptor 
and an MHC molecule. 



The binding of an MHC molecule to an antigenic peptide 
does not have the fine specificity of the interaction between an 
antibody and its epitope. Instead, a given MHC molecule can 
selectively bind a variety of different peptides. For example, the 
class II MHC molecule designated IA can bind peptides from 
ovalbumin (residues 323-339), hemagglutinin (residues 130— 
142), and lambda repressor (residues 12-26). Studies revealing 
structural features, or motifs, common to different peptides 
that bind to a single MHC molecule are described in Chapter 7. 

Antigen processing is required to generate peptides that in- 
teract specifically with MHC molecules. As mentioned in 
Chapter 1, endogenous and exogenous antigens are usually 
processed by different intracellular pathways (see Figure 
1-9). Endogenous antigens are processed into peptides 
within the cytoplasm, while exogenous antigens are 
processed by the endocytic pathway. The details of antigen 
processing and presentation are described in Chapter 8. 

Epitopes recognized by T cells are often internal. T cells tend 
to recognize internal peptides that are exposed by processing 
within antigen-presenting cells or altered self-cells. J. Roth- 
bard analyzed the tertiary conformation of hen egg-white 
lysozyme and sperm whale myoglobin to determine which 
amino acids protruded from the natural molecule. He then 
mapped the major T-cell epitopes for both proteins and 
found that, in each case, the T-cell epitopes tended to be on 
the "inside" of the protein molecule (Figure 3-9). 



Haptens and the Study 
of Antigenicity 

The pioneering work of Karl Landsteiner in the 1920s and 
1930s created a simple, chemically defined system for study- 
ing the binding of an individual antibody to a unique epitope 



T-cell epitopes of hen egg-white lysozyme 
34 45 51 61 78 93 




Residue number 



Experimental evidence 
internal peptides of antigens. This plo 
of amino acid residues in the tertiary c 
lysozyme. The known T-cell epitopes ir 
bars at the top. Notice that, in genera: 



shows the relative protrusior 
jnformation of hen egg-white 
HELare indicated by the blue 



correspond to the T-cell epitopes exhibit 



II protrusio 



contrast, note that the B-cell epitope consisting of residues 64-80, 
which form a conformational determinant in native HELthat is rec- 
ognized by antibody (see Figure 3-/), exhibit greater overall protru- 
sion. [From J. Rothbard et al., 1987, Mod. Trends Hum. Leuk., vol. 7.] 



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: al . / Immunology 5e : 



on a complex protein antigen. Landsteiner employed various 
haptens, small organic molecules that are antigenic but not 
immunogenic. Chemical coupling of a hapten to a large pro- 
tein, called a carrier, yields an immunogenic hapten-carrier 
conjugate. Animals immunized with such a conjugate pro- 
duce antibodies specific for (1) the hapten determinant, (2) 
unaltered epitopes on the carrier protein, and (3) new epi- 
topes formed by combined parts of both the hapten and car- 
rier (Figure 3-10). By itself, a hapten cannot function as an 
immunogenic epitope. But when multiple molecules of a sin- 
gle hapten are coupled to a carrier protein (or nonimmuno- 
genic homopolymer), the hapten becomes accessible to the 
immune system and can function as an immunogen. 

The beauty of the hapten-carrier system is that it provides 
immunologists with a chemically defined determinant that 
can be subtly modified by chemical means to determine the 
effect of various chemical structures on immune specificity. 
In his studies, Landsteiner immunized rabbits with a hapten- 
carrier conjugate and then tested the reactivity of the rabbit's 
immune sera with that hapten and with closely related hap- 
tens coupled to a different carrier protein. He was thus able to 
measure, specifically, the reaction of the antihapten antibod- 
ies in the immune serum and not that of antibodies to the 




Antibodies to conjugate 
of hapten and carrier 



on with: 


Antibodies formed: 


Hapten (DNP) 


None 


Protein carrier (BSA) 


Anti-BSA 


Hapten-carrier 


Anti-DNP (major) 


conjugate (DNP-BSA) 


Anti-BSA (minor) 




Anti-DNP/BSA (minor) 



' A hapten-carrier conjugate contains multiple copies 
of the hapten — a small nonimmunogenic organic compound such 
as dinitrophenol (DNP)— chemically linked to a large protein carrier 
such as bovine serum albumin (BSA). Immunization with DNP 
alone elicits no anti-DNP antibodies, but immunization with DNP- 
BSA elicits three types of antibodies. Of these, anti-DNP antibody is 
predominant, indicating that in this case the hapten is the immuno- 
dominant epitope in a hapten-carrier conjugate, as it often is in such 
conjugates. 



original carrier epitopes. Landsteiner tested whether an anti- 
hapten antibody could bind to other haptens having a 
slightly different chemical structure. If a reaction occurred, it 
was called a cross-reaction. By observing which hapten 
modifications prevented or permitted cross-reactions, Land- 
steiner was able to gain insight into the specificity of antigen- 
antibody interactions. 

Using various derivatives of aminobenzene as haptens, 
Landsteiner found that the overall configuration of a hapten 
plays a major role in determining whether it can react with 
a given antibody. For example, antiserum from rabbits im- 
munized with aminobenzene or one of its carboxyl deriva- 
tives (o-aminobenzoic acid, m-aminobenzoic acid, or p- 
aminobenzoic acid) coupled to a carrier protein reacted only 
with the original immunizing hapten and did not cross-react 
with any of the other haptens (Table 3-6). In contrast, if the 
overall configuration of the hapten was kept the same and 
the hapten was modified in the para position with various 
nonionic derivatives, then the antisera showed various de- 
grees of cross-reactivity. Landsteiner 's work not only demon- 
strated the specificity of the immune system, but also demon- 
strated the enormous diversity of epitopes that the immune 
system is capable of recognizing. 

Many biologically important substances, including drugs, 
peptide hormones, and steroid hormones, can function as 
haptens. Conjugates of these haptens with large protein car- 
riers can be used to produce hapten-specific antibodies. 
These antibodies are useful for measuring the presence of 
various substances in the body. For instance, the original 
home pregnancy test kit employed antihapten antibodies to 
determine whether a woman's urine contained human chori- 
onic gonadotropin (HCG), which is a sign of pregnancy. 
However, as shown in the Clinical Focus, the formation of 
drug-protein conjugates in the body can produce drug aller- 
gies that may be life-threatening. 



Pattern-Recognition Receptors 

The receptors of adaptive and innate immunity differ. Anti- 
bodies and T-cell receptors, the receptors of adaptive immu- 
nity, recognize details of molecular structure and can 
discriminate with exquisite specificity between antigens fea- 
turing only slight structural differences. The receptors of in- 
nate immunity recognize broad structural motifs that are 
highly conserved within microbial species but are generally 
absent from the host. Because they recognize particular over- 
all molecular patterns, such receptors are called pattern- 
recognition receptors (PRRs). Patterns recognized by this 
type of receptor include combinations of sugars, certain pro- 
teins, particular lipid-bearing molecules, and some nucleic 
acid motifs. Typically, the ability of pattern-recognition 
receptors to distinguish between self and nonself is perfect 
because the molecular pattern targeted by the receptor is 
produced only by the pathogen and never by the host. This 
contrasts sharply with the occasional recognition of self 



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Generation of B-Cell and T-Cell Respor 



REACTIVITY \X 



&" o 



NH, 

6 



Antiserum against Aminobenzene (aniline) o-Aminobenzoic acid m-Aminobenzoic acid p-Aminobenzoic acid 

Aminobenzene +000 

o-Aminobenzoic acid + 00 

m-Aminobenzoic acid + 

p-Aminobenzoic acid + 



antigens by receptors of adaptive immunity, which can lead 
to autoimmune disorders. Like antibodies and T-cell recep- 
tors, pattern-recognition receptors are proteins. However, 
the genes that encode PRRs are present in the germline of the 
organism. In contrast, the genes that encode the enormous 
diversity of antibodies and TCRs are not present in the 
germline. They are generated by an extraordinary process of 
genetic recombination that is discussed in Chapter 5. 

Many different pattern-recognition receptors have been 
identified and several examples appear in Table 3-7. Some are 
present in the bloodstream and tissue fluids as soluble circu- 
lating proteins and others are on the membrane of cells such 
as macrophages, neutrophils, and dendritic cells. Mannose- 
binding lectin (MBL) and C-reactive protein (CRP) are solu- 
ble pattern receptors that bind to microbial surfaces and 
promote their opsonization. Both of these receptors also 
have the ability to activate the complement system when they 
are bound to the surface of microbes, thereby making the 
invader a likely target of complement-mediated lysis. Yet 
another soluble receptor of the innate immune system, 
lipopolysaccharide-binding protein, is an important part 
of the system that recognizes and signals a response to 
lipopolysaccharide, a component of the outer cell wall of 
gram-negative bacteria. 

Pattern-recognition receptors found on the cell mem- 
brane include scavenger receptors and the toll-like receptors. 
Scavenger receptors (SRs) are present on macrophages and 
many types of dendritic cells, and are involved in the binding 
and internalization of gram-positive and gram-negative bac- 
teria, as well as the phagocytosis of apoptotic host cells. The 
exact roles and mechanisms of action of the many types of 
scavenger receptors known to date are under active investiga- 
tion. The toll-like receptors (TLRs) are important in recog- 
nizing many microbial patterns. This family of proteins is 



ancient — toll-like receptors mediate the recognition and 
generation of defensive responses to pathogens in organisms 
as widely separated in evolutionary history as humans and 
flies. Typically, signals transduced through the TLRs cause 
transcriptional activation and the synthesis and secretion of 
cytokines, which promote inflammatory responses that 
bring macrophages and neutrophils to sites of inflammation. 



Lipoproteins LPS (Gram-negative) Flagellin CpG DNA 

Lipoarabinomannan Taxol (Plant) 

LPS {Leptospira) F protein (RS virus) 

LPS {P. gingivalis) hsp60 (Host) 

PGN (Gram-positive) Fibronectin (Host) 

Zymosan (Yeast) 

GPI anchor (/: cruzi) 



|| lflMD-2 | | 

TLR2 TLR6 TLR4 W- 1 TLR5 TLR9 1 

— ii — i — |— r 



Bl Locatioi 
ceptors. Many pattern-rf 






ofsc 



; pattern-recognition re- 
n receptors are extracellular and tar- 
get microbes or microbial components in the bloodstream and 
tissue fluids, causing their lysis or marking them for removal by 
phagocytes. Other pattern-recognition receptors are present on the 
cell membrane and bind to a broad variety of microbes or microbial 
products. Engagement of these receptors triggers signaling path- 
ways that promote inflammation or, in the case of the scavenger re- 
ceptors, phagocytosis or endocytosis. dsRNA = double stranded 
RNA; LPS = lipopolysaccharide. [S. Akira et al., 2001, Nature Im- 
munology 2:675.] 



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' Immunology 5e: 



Characteristic 

Specificity 
Self/nonself 






jnity 



Specific for conserved 

molecular patterns or types 
Perfect: evolutionary selected 

:o distinguish phylogenetic 

. Never recognizes 



differe 



self. 



Adaptive immunity 

Specific for details of antigen 

Excellent: but imperfect. 
Occasional reaction with 
self antigens 



RECEPTORS OF THE ADAPTIVE IMMUNE SYSTEM 



Receptor 
(location) 



Antibody 
(B-cell membrane, 
blood, tissue fluids) 



Target 
(source) 



Specific components of 
pathogen 



Proteins or certain lipids of 
pathogen 



Induction of pathogen- 
specific humoral and cell- 
mediated immunity 



Complement 
(bloodstream, 
tissue fluids) 

Mannose-binding lectin (MBL) 
(bloodstream, tissue fluids) 

C-reactive protein (CRP) 
(bloodstream, tissue fluids) 

LPS-binding protein (LBP) 
(bloodstream, tissue fluids) 

TLR2 
(cell membrane) 



TLR4 
(cell membrane) 



Scavenger receptors (many) 
(cell membrane) 



RECEPTORS OF THE INNATE II 



Microbial cell-v 
components 



microbial carbohydrates 

Phosphatidylcholine 
(microbial membranes) 

Bacterial lipopolysaccharide 
(LPS) 

Cell-wall components of gram-positive 
bacteria, LPS*. Yeast cell-wall component 
(zymosan) 

Double-stranded RNA (dsRNA) 
(replication of many RNA viruses) 



Flagellin 
and gram-negative bacteria) 



Many targets; gram-positive and gram- 
negative bacteria, apoptotic host cells 






Complement 
opsonization 

Delivery to cell-membrane LPS receptor 
(TLR-CD14-MD-2 complex*) 

Attracts phagocytes, activates macrophages, 
dendritic cells. Induces secretion of 
several cytokines 

Induces production of interferon, 
an antiviral cytokine 

Attracts phagocytes, activates macrophages, 
dendritic cells. Induces secretion of 
several cytokines 

Attracts phagocytes, activates macrophages, 
dendritic cells. Induces secretion of 
several cytokines 

Attracts phagocytes, macrophages, 
dendritic cells. Induces secretion of 

Induces phagocytosis or endocytosis 



aTLR (usually TLR4). 



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CLINICAL FOCUS 



Drug Allergies — When 
Medicines Become 
Immunogens 



SincG Worl warn 

penicillin has been used to successfully 
treat a wide variety of bacterial infec- 
tions. However, the penicillin family of 
antibiotics is not without drawbacks. 
One is the role of penicillins and other 
antibiotics in the evolution of antibiotic- 
resistant bacterial strains. Another is 
their capacity to induce allergic reactions 
in some patients. Penicillin and its rela- 
tives are responsible for most of the 
recorded allergic reactions to drugs and 
97% of the deaths caused each year by 
drug allergies. 

Allergies to penicillin and other drugs 
can be induced by small doses and are 
not consequences of the pharmacologi- 
cal or physiological effects of the drugs. 
An allergic response usually occurs 
about a week or so after the patient's first 
exposure to the agent, with typically mild 
symptoms often including hives, fever, 
swelling of lymph nodes, and occasion- 



ally an arthritis-like discomfort. Subse- 
quent treatments with the drug usually 
cause much more rapid and often more 
severe reactions. Within minutes the 
throat and eyelids may swell. Crave dan- 
ger arises if these symptoms progress to 
anaphylaxis, a physiological collapse that 
often involves the respiratory, circulatory, 
and digestive systems. Hives, vomiting, 
abdominal pain, and diarrhea may be a 
preamble to respiratory and circulatory 
problems that are life threatening. 
Wheezing and shortness of breath may 
be accompanied by swelling of the larynx 
and epiglottis that can block airflow, and 
a profound drop in blood pressure 
causes shock, frequently accompanied 
by weakened heart contractions. 

The treatment of choice for anaphy- 
laxis is injection of the drug epinephrine 
(adrenaline), which can reverse the 
body's slide into deep anaphylaxis by 
raising blood pressure, easing constric- 
tion of the air passages, and inhibiting 



the release from mast cells and ba- 
sophils of the agents that induce ana- 
phylaxis. Other drugs may be used to 
raise the low blood pressure, strengthen 
heart contractions, and expand the 
blocked airways. After a case of drug-in- 
duced anaphylaxis, affected individuals 
are advised to carry a notice warning 
future healthcare providers of the drug 
allergy. 

Most drugs, including penicillin, are 
low-molecular-weight compounds that 
cannot induce immune responses un- 
less they are conjugated with a larger 
molecule. Intensive investigation of al- 
lergy to penicillin has provided critical in- 
sight into the basis of allergic reactions 
to this and other drugs. As shown in the 
accompanying figure, penicillin can react 
with proteins to form a penicilloyl-pro- 
tein derivative. The penicilloyl-protein 
behaves as a hapten-carrier conjugate, 
with the penicilloyl group acting as a 
haptenic epitope. This epitope is readily 
recognized by the immune system, and 
antibodies are produced against it. Some 
individuals respond to penicillin by pro- 
ducing significant amounts of a type of 
antibody known as immunoglobulin E 
(IgE). Once generated, these IgE anti- 
bodies are dispersed throughout the 
body and are bound by IgE receptors on 
the surfaces of mast cells and basophils, 



TLR signaling can also result in the recruitment and activa- 
tion of macrophages, NK cells, and dendritic cells, key agents 
in the presentation of antigen to T cells. The links to T cells 
and cytokine release shows the intimate relationship between 
innate and adaptive responses. 

A search of the human genome has uncovered 10 TLRs, 
and the functions of six members of this PRR family have 
been determined. TLR2, often with the collaboration of 
TLR6, binds a wide variety of molecular classes found in mi- 
crobes, including peptidoglycans, zymosans, and bacterial 
lipopeptides. TLR4 is the key receptor for most bacterial 
lipopolysaccharides, although TLR2 also binds some vari- 
eties of LPS. The binding of LPS by either of these TLRs is 
complex and involves the participation of three additional 
proteins, one of which is the lipopolysaccharide-binding 
protein mentioned above, abbreviated LBP The first step in 
the process is the binding of LPS by circulating LBP, which 



then delivers it to a complex of TLR4 (or TLR2) with two ad- 
ditional proteins, CD 14 and MD2. The engagement of LPS 
by this complex causes its TLR component to initiate a sig- 
nal-transduction process that can produce a cellular re- 
sponse. Another family member, TLR5, recognizes flagellin, 
the major structural component of bacterial flagella. TLR3 
recognizes the double-stranded RNA (dsRNA) that appears 
after infection by RNA viruses. As shown in Table 3-7, 
dsRNA is also recognized by dsRNA-activated kinase. Finally, 
TLR9 recognizes and initiates a response to CpG (unmethy- 
lated cytosine linked to guanine) sequences. These sequences 
are represented in abundance in microbial sequences but are 
much less common in mammalian sequences. Table 3-7 
summarizes the receptors of adaptive immunity and lists 
many pattern-recognition receptors of innate immunity. The 
microbial targets and physiological sites of many PRRs are 
shown in Figure 3-11. 



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Penicillin 




Penicillenic acid 




'l 


Isomerization 




o=c 




— > O — C 




HN 






N SH 2 




1 H/\ 






1 H | 




HC — C 

1 




C = C C(CH,>7 

| 




C — N CH 






— C N — CH 




O COOH 




O COOH 


When nucleophiles such as amino groups or hydroxyl groups 


Protein 






Same or 


are present on soluble proteins or on the membrane of cells, 






different 


they can react with penicillin and its relatives to form covalent 


Penicilloyl-protein 






protein 


linkages between host macromolecular structures and the 


R 






drug. This is illustrated by the reaction of the free amino 






Reaction of 


group of a lysine residue with penicillin (or with its sponta- 


| 




isomeric structures 


neously forming isomeric compounds, such as penicillenic 


HI ? S 




> proteins 


acid) to produce protein-drug or cell-surface-drug derivatives. 


1 H/\ 




produces a 


Such adducts are the major immunogenic species that elicit 


HC — C ( 

i-4-i 1 1 


variety of major 


immune responses to this antibiotic. However, as indicated, 


HO-FcT-N CH 




and minor 


other hapten-carrier conjugates of somewhat different struc- 


N COOP 


determinants 


ture are also formed and, because of their structural similarity, 


L^lJ 






can also induce immune responses to penicillin. [Adapted 


(CH 2 ) 4 






from N. F. Adkinson, 1995, in Manual of Clinical Laboratory 


H I 
N — C — C Prote n 




Immunology, N. Rose et al., eds., American Society for 


H || 






Microbiology, Washington, D.C.] 


O 






where they can remain for a long time. If Penicillin is 


not the only drug against 


tives. When th 


s happens, there is a pos- 


a person with penicillin-specific IgE anti- which patients 


can develop allergies. 


sibility that the 


immune system will pro- 


body bound to mast cells is subse- Others include 


streptomycin, aspirin, the 


duce an anti- 


tapten response to the 


quently treated with penicillin, there may so-called "sulfa 


-drugs" such as the sul- 


drug, just as v. 


ith penicillin. Drugs (and 


be an allergic reaction. In fact, between l fonamides, son 


ne anesthetics (e.g., suc- 


their metabolites) that are incapable of 


and 5 percent of people treated with cinyl choline), 


and some opiates. All of 


forming drug-protein conjugates rarely 


penicillin develop some degree of allergy these small m 


olecules first react with 


elicit allergic reactions. 


to it. proteins to form drug-protein deriva- 







SUMMARY 

■ All immunogens are antigens but not all antigens are im- 
munogens. 

■ Immunogenicity is determined by many factors including 
foreignness, molecular size, chemical composition, com- 
plexity, dose, susceptibility to antigen processing and pre- 
sentation, the genotype of the recipient animal (in 
particular, its MHC genes), route of administration, and 
adjuvants. 

■ The sizes of B-cell epitopes range widely. Some are quite 
small (e.g., small peptides or small organic molecules), 
and are often bound in narrow grooves or deep pockets of 
the antibody. Protein B-cell epitopes are much larger and 
interact with a larger, flatter complementary surface on 
the antibody molecule. 



■ T-cell epitopes are generated by antigen processing, which 
fragments protein into small peptides that combine with 
class I or class II MHC molecules to form peptide-MHC 
complexes that are displayed on the surface of cells. T-cell 
activation requires the formation of a ternary complex 
between a T cell's TCR and peptide-MHC on antigen- 
presenting or altered self cells. 

■ Haptens are small molecules that can bind to antibodies 
but cannot by themselves induce an immune response. 
However, the conjugate formed by coupling a hapten to a 
large carrier protein is immunogenic and elicits produc- 
tion of anti-hapten antibodies when injected into an ani- 
mal. Such injections also produce anti-carrier and anti- 
hapten/carrier antibodies as well. 

■ In the body, the formation of hapten-carrier conjugates is 
the basis of allergic responses to drugs such as penicillin. 



Go to www.whfreeman.com 
Review and quiz of key tern- 



© S 



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' Immunology 5e: 



Generation of B-Cell and T-Cell Respor 



■ The innate immune system uses pattern-recognition re- 
ceptors to recognize and respond to broad structural mo- 
tifs that are highly conserved within microbial species but 
are generally absent from the host. 

References 

Berzofsky, J. A., and J. J. Berkower. 1999. Immunogenicity and 
antigen structure. In Fundamental Immunology, 4th ed., 
W. E. Paul, ed., Lippincott-Raven, Philadelphia. 

Dale, D., and D. Federman, eds. 1997. Drug allergy. In Scientific 
American Medicine. Chapter VIII, Hypersensitivity and allergy, 
p. 27. 

Demotz, S., H. M. Grey, E. Appella, and A. Sette. 1989. Charac- 
terization of a naturally processed MHC class Il-restricted T- 
cell determinant of hen egg lysozyme. Nature 342:682. 

Grey, H. M., A. Sette, and S. Buus. 1989. How T cells see antigen. 
Sci. Am. 261(5):56. 

Landsteiner, K. 1945. The Specificity of Serological Reactions. 
Harvard University Press, Cambridge, Massachusetts. 

Laver, W. G., G. M. Air, R. G. Webster, and S. J. Smith-Gill. 1990. 
Epitopes on protein antigens: misconceptions and realities. 
Cell 61:553. 

Peiser, L., S. Mukhopadhyay, and S. Gordon. 2002. Scavenger re- 
ceptors in innate immunity. Curr. Opin. Immunol. 14:123. 

Stanfield, R. L., and I. A. Wilson. 1995. Protein-peptide interac- 
tions. Curr. Opin. Struc. Biol. 5:103. 

Tainer, J. A., et al. 1985. The atomic mobility component of pro- 
tein antigenicity. Annu. Rev. Immunol. 3:501. 

Underhill, D. M., and A. Ozinsky. 2002. Toll-like receptors: key 
mediators of microbe detection. Curr. Opin. Immunol. 14:103. 



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Study Questions 



Clinical Focus Question Consider the following situations 
and provide a likely diagnosis or appropriate response. 



a. Six hours after receiving a dose of penicillin, a young child 
who has never been treated with penicillin develops a case 
of hives and diarrhea. The parents report the illness and 
ask if it might be an allergic reaction to penicillin. 

b. A patient who has never taken sulfonamides but is known 
to be highly allergic to penicillin develops a bladder infec- 
tion that is best treated with a "sulfa" drug. The patient 
wonders if "sulfa" drugs should be avoided. 

c. A student who is unaware that he had developed a signifi- 
cant allergy to penicillin received an injection of the an- 
tibiotic and within minutes experienced severe respiratory 
distress and a drop in blood pressure. An alert intern 
administered epinephrine and the patient's condition 
improved quickly. Frightened but impressed by the 
effectiveness of the treatment, he asked the intern why the 
shot of adrenaline made him feel better. 

d. A pet owner asks whether the same mechanism that causes 
his allergy to penicillin could also be responsible for his 
dog's development of a similar allergy to the drug. (Please 
go beyond yes or no.) 

1 . Indicate whether each of the following statements is true or 
false. If you think a statement is false, explain why. 

a. Most antigens induce a response from more than one 

b. A large protein antigen generally can combine with many 
different antibody molecules. 

c. A hapten can stimulate antibody formation but cannot 
combine with antibody molecules. 

d. MHC genes play a major role in determining the degree of 
immune responsiveness to an antigen. 

e. T-cell epitopes tend to be accessible amino acid residues 
that can combine with the T-cell receptor. 

f. Many B-cell epitopes are nonsequential amino acids 
brought together by the tertiary conformation of a protein 
antigen. 

g. Both T H and T c cells recognize antigen that has been 
processed and presented with an MHC molecule. 

h. Each MHC molecule binds a unique peptide. 

i. All antigens are also immunogens. 

j. Antibodies can bind hydrophilic or hydrophobic com- 
pounds, but T-cell receptors can only bind peptide-MHC 
complexes. 

2. What would be the likely outcome of each of the develop- 
ments indicated below. Please be as specific as you can. 

a. An individual is born with a mutation in C-reactive pro- 
tein that enables it to recognize phospholipids in both bac- 
terial and mammalian cell membranes. 

b. A group of mice in which the CD1 family has been 
"knocked out" are immunized with Mycobacterium tuber- 
culosis. Spleen cells from these mice are isolated and di- 
vided into two batches. One batch is treated with a lipid 
extract of the bacteria and a second batch is treated with a 
protein derived from the bacteria known as purified pro- 
tein derivative (PPD). 

3. Two vaccines are described below. Would you expect either or 
both of them to activate T c cells? Explain your answer. 

a. A UV-inactivated ("killed") viral preparation that has re- 
tained its antigenic properties but cannot replicate. 



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' Immunology 5e: 



b. An attenuated viral preparation that has low virulence but 
can still replicate within host cells. 
4. For each pair of antigens listed below, indicate which is likely 
to be more immunogenic. Explain your answer. 

a. Native bovine serum albumin (BSA) 
Heat-denatured BSA 

b. Hen egg-white lysozyme (HEL) 
Hen collagen 

c. A protein with a molecular weight of 30,000 
A protein with a molecular weight of 150,000 

d. BSA in Freund's complete adjuvant 
BSA in Freund's incomplete adjuvant 



e. Carriers include small molecules such as dinitrophenc 
and penicillenic acid (derived from penicillin). 

6. For each of the following statements, indicate whether it 
true only of B-cell epitopes (B), only of T-cell epitopes (T),c 
both types of epitopes (BT) within a large antigen. 



a. They almost always consist 

b. They generally are located ii 






5. Indicate which of the following 



regarding hapte 



a. Haptens are large protein molecules such as BSA. 

b. When a hapten-carrier complex containing multiple hap- 
ten molecules is injected into an animal, most of the in- 
duced antibodies are specific for the hapten. 

c. Carriers are needed only if one wants to elicit a cell-medi- 
ated response. 

d. It is necessary to immunize with a hapten-carrier complex 
in order to obtain antibodies directed against the hapten. 



c. They generally are located on the surface of a protein anti- 
gen. 

d. They lose their immunogenicity when a protein antigen is 
denatured by heat. 

e. Immunodominant epitopes are determined in part by the 
MHC molecules expressed by an individual. 

f. They generally arise from proteins. 

g. Multiple different epitopes may occur in the same antigen. 
h. Their immunogenicity may depend on the three-dimen- 
sional structure of the antigen. 

i. The immune response to them may be enhanced by co- 
of Freund's complete adjuvant. 



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12:03 PM Page 76 mac76 mac76:385_i 



limited 



Antibodies: 

Structure and Function 



/NTIBODIES ARE THE ANTIGEN-BINDING PROTEINS 
present on the B-cell membrane and secreted by 
plasma cells. Membrane-bound antibody con- 
fers antigenic specificity on B cells; antigen-specific prolifer- 
ation of B-cell clones is elicted by the interaction of 
membrane antibody with antigen. Secreted antibodies cir- 
culate in the blood, where they serve as the effectors of hu- 
moral immunity by searching out and neutralizing antigens 
or marking them for elimination. All antibodies sha 
tural features, bind to antigen, and participate in £ 
number of effector functions. 

The antibodies produced in response to a particular 
gen are heterogeneous. Most antigens are complex and 
tain many different antigenic determinants, and the 
system usually responds by producing antibodies to several 
epitopes on the antigen. This response requires the recruit- 
ment of several clones of B cells. Their outputs are mono- 
clonal antibodies, each of which specifically binds a single 
antigenic determinant. Together, these monoclonal antibod- 
ies make up the polyclonal and heterogeneous serum anti- 
body response tc 



Basic Structure of Antibodies 

Blood can be separated in a centrifuge into a fluid and a cel- 
lular fraction. The fluid fraction is the plasma and the cellu- 
lar fraction contains red blood cells, leukocytes, and 
platelets. Plasma contains all of the soluble small molecules 
and macromolecules of blood, including fibrin and other 
proteins required for the formation of blood clots. If the 
blood or plasma is allowed to clot, the fluid phase that re- 
mains is called serum. It has been known since the turn of 
the century that antibodies reside in the serum. The first 
evidence that antibodies were contained in particular 
serum protein fractions came from a classic experiment by 
A. Tiselius and E. A. Kabat, in 1939. They immunized rabbits 
with the protein ovalbumin (the albumin of egg whites) and 
then divided the immunized rabbits' serum into two 
aliquots. Electrophoresis of one serum aliquot revealed four 
peaks corresponding to albumin and the alpha (a), beta ((}), 
and gamma (7) globulins. The other serum aliquot was re- 
acted with ovalbumin, and the precipitate that formed was 
removed; the remaining serum proteins, which did not react 
with the antigen, were then electrophoresed. A comparison 
rofiles of these two serum aliquots 
a significant drop in the 7-globulin 



chapter 4 




■ Basic Structure of Antibodies 

■ Obstacles to Antibody Sequencing 

■ Immunoglobulin Fine Structure 

■ Antibody-Mediated Effector Functions 

■ Antibody Classes and Biological Activities 

■ Antigenic Determinants on Immunoglobulins 

■ The B-Cell Receptor 

■ The Immunoglobulin Superfamily 

■ Monoclonal Antibodies 



of the electrophoi 
revealed that there v 



peak in the aliquot that had been reacted with antigen (Fig- 
ure 4- 1 ). Thus, the 7-globulin fraction was identified as con- 
taining serum antibodies, which were called immunoglob- 
ulins, to distinguish them from any other proteins that might 
be contained in the 7-globulin fraction. The early experi- 
ments of Kabat and Tiselius resolved serum proteins into 
three major nonalbumin peaks — a, (3 and 7. We now know 
that although immunoglobulin G (IgG), the main class of 
antibody molecules, is indeed mostly found in the 7-globulin 
fraction, significant amounts of it and other important 
classes of antibody molecules are found in the a and the (3 
fractions of serum. 

Antibodies Are Heterodimers 



Antibody molecules have a common structure of four 
peptide chains (Figure 4-2). This structure consists of two 
identical light (L) chains, polypeptides of about 25,000 
molecular weight, and two identical heavy (H) chains, larger 



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fe 















- 




Albumin 
















/A\ , 




Clob lln- 


. 












! 














Y 


'i 
















$ 


- 








a 


P 






" 




/.■ 









Migration distance 

in the -y-globulin fraction of serum proteins. After rabbits were im- 
munized with ovalbumin (OVA), their antisera were pooled and elec- 
trophoresed, which separated the serum proteins according to their 
electric charge and mass. The blue line shows the electrophoretic 
pattern of untreated antiserum. The black line shows the pattern of 
antiserum that was incubated with OVA to remove anti-OVA anti- 
body and then electrophoresed. [Adapted from A. Tiselius and E. A. 
Kabat, 1939, J. Exp. Med. 69:119, with copyright permission of the 
Rockefeller University Press.] 



polypeptides of molecular weight 50,000 or more. Like the 
antibody molecules they constitute, H and L chains are also 
called immunoglobulins. Each light chain is bound to a 
heavy chain by a disulfide bond, and by such noncovalent in- 
teractions as salt linkages, hydrogen bonds, and hydrophobic 
bonds, to form a heterodimer (H-L). Similar noncovalent in- 
teractions and disulfide bridges link the two identical heavy 
and light (H-L) chain combinations to each other to form the 
basic four-chain (H-L) 2 antibody structure, a dimer of 
dimers. As we shall see, the exact number and precise posi- 
tions of these interchain disulfide bonds differs among anti- 
body classes and subclasses. 

The first 110 or so amino acids of the amino-terminal re- 
gion of a light or heavy chain varies greatly among antibodies 
of different specificity. These segments of highly variable se- 
quence are called Vregions:V L in light chains and V H in heavy. 
All of the differences in specificity displayed by different anti- 
bodies can be traced to differences in the amino acid se- 
quences of V regions. In fact, most of the differences among 
antibodies fall within areas of the V regions called comple- 
mentarity-determining regions (CDRs), and it is these CDRs, 
on both light and heavy chains, that constitute the antigen- 
binding site of the antibody molecule. By contrast, within the 
same antibody class, far fewer differences are seen when one 
compares sequences throughout the rest of the molecule. The 
regions of relatively constant sequence beyond the variable re- 
gions have been dubbed C regions, C L on the light chain and 



C H on the heavy chain. Antibodies are glycoproteins; with few 
exceptions, the sites of attachment for carbohydrates are re- 
stricted to the constant region. We do not completely under- 
stand the role played by glycosylation of antibodies, but it 
probably increases the solubility of the molecules. Inappro- 
priate glycosylation, or its absence, affects the rate at which 
antibodies are cleared from the serum, and decreases the effi- 
ciency of interaction between antibody and the complement 
system and between antibodies and Fc receptors. 

Chemical and Enzymatic Methods Revealed 
Basic Antibody Structure 

Our knowledge of basic antibody structure was derived from 
a variety of experimental observations. When the -y-globulin 
fraction of serum is separated into high- and low-molecular- 
weight fractions, antibodies of around 150,000-MW, des- 
ignated as immunoglobulin G (IgG) are found in the low- 
molecular-weight fraction. In a key experiment, brief diges- 
tion of IgG with the enzyme papain produced three frag- 
ments, two of which were identical fragments and a third that 
was quite different (Figure 4-3). The two identical fragments 



Heavy chain Light chain 
u,y,a,5, or e k or X 




coo- coo- 



Schematic diagram of structure of immunoglobulins 
derived from amino acid sequencing studies. Each heavy and light 
chain in an immunoglobulin molecule contains an amino-terminal 
variable (V) region (aqua and tan, respectively) that consists of 100- 
1 10 amino acids and differs from one antibody to the next. The re- 
mainder of each chain in the molecule— the constant (C) regions 
(purple and red)— exhibits limited variation that defines the two 
ight-chain subtypes and the five heavy-chain subclasses. Some 
heavy chains (-y, 8, and a) also contain a proline-rich hinge region 
(black). The amino-terminal portions, corresponding to the V re- 
gions, bind to antigen; effector functions are mediated by the other 
domains. The u, and e heavy chains, which lack a hinge region, con- 
tain an additional domain in the middle of the molecule. 



whfreeman.cc 



mnology 



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Generation of B-Cell and T-Cell Respor 




* Prototype structure of IgC, showing chain stn 
and interchain disulfide bonds. The fragments produced by vi 



3 indicated. Light (L) chai 



(each with a MW of 45,000), had antigen-binding activity 
and were called Fab fragments ("fragment, antigen bind- 
ing"). The other fragment (MW of 50,000) had no antigen- 
binding activity at all. Because it was found to crystallize 
during cold storage, it was called the Fc fragment ("frag- 
ment, crystallizable"). Digestion with pepsin, a different pro- 
teolytic enzyme, also demonstrated that the antigen-binding 
properties of an antibody can be separated from the rest of 
the molecule. Pepsin digestion generated a single 100,000- 
MW fragment composed of two Fab-like fragments desig- 
nated the F(ab')2 fragment, which binds antigen. The Fc 
fragment was not recovered from pepsin digestion because it 
had been digested into multiple fragments. 

A key observation in deducing the multichain structure of 
IgG was made when the molecule was subjected to mercap- 
toethanol reduction and alkylation, a chemical treatment 
that irreversibly cleaves disulfide bonds. If the sample is chro- 
matographed on a column that separates molecules by size 
following cleavage of disulfide bonds, it is clear that the intact 
150,000-MW IgG molecule is, in fact, composed of subunits. 
Each IgG molecule contains two 50,000-MW polypeptide 
chains, designated as heavy (H) chains, and two 25,000-MW 
chains, designated as light (L) chains (see Figure 4-3). 

Antibodies themselves were used to determine how the 
enzyme digestion products — Fab, F(ab') 2) and Fc — were re- 
lated to the heavy-chain and light-chain reduction products. 



This question was answered by using antisera from goats that 
had been immunized with either the Fab fragments or the Fc 
fragments of rabbit IgG. The antibody to the Fab fragment 
could react with both the H and the L chains, whereas anti- 
body to the Fc fragment reacted only with the H chain. These 
to the conclusion that the Fab fragment 
; of portions of a heavy and a light chain and that Fc 
ily heavy-chain components. From these results, 
and those mentioned above, the structure of IgG shown in 
Figure 4-3 was deduced. According to this model, the IgG 
molecule consists of two identical H chains and two identical 
L chains, which are linked by disulfide bridges. The enzyme 
papain cleaves just above the interchain disulfide bonds link- 
ing the heavy chains, whereas the enzyme pepsin cleaves just 
below these bonds, so that the two proteolytic enzymes gen- 
erate different digestion products. Mercaptoethanol reduc- 
tion and alkylation allow separation of the individual heavy 
and light chains. 



Obstacles to Antibody Sequencing 

Initial attempts to determine the amino acid sequence of the 
heavy and light chains of antibody were hindered because in- 
sufficient amounts of homogeneous protein were available. 
Although the basic structure and chemical properties of differ- 



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9/6/02 9:02 PM Page 79 mac85 Mac 85:365_E 



ent antibodies are similar, their antigen-binding specificities, 
and therefore their exact amino acid sequences, are very differ- 
ent. The population of antibodies in the serum 7-globulin 
fraction consists of a heterogeneous spectrum of antibodies. 
Even if immunization is done with a hapten-carrier conjugate, 
the antibodies formed just to the hapten alone are heteroge- 
neous: they recognize different epitopes of the hapten and 
have different binding affinities. This heterogeneity of serum 
antibodies made them unsuitable for sequencing studies. 

Pure Immunoglobulin Obtained from 
Multiple Myeloma Patients Made 
Sequencing Possible 

Sequencing analysis finally became feasible with the discov- 
ery of multiple myeloma, a cancer of antibody-producing 
plasma cells. The plasma cells in a normal individual are end- 
stage cells that secrete a single molecular species of antibody 
for a limited period of time and then die. In contrast, a clone 
of plasma cells in an individual with multiple myeloma has 
escaped normal controls on their life span and proliferation 
and are not end- stage cells; rather, they divide over and over 
in an unregulated way without requiring any activation by 
antigen to induce proliferation. Although such a cancerous 
plasma cell, called a myeloma cell, has been transformed, its 
protein-synthesizing machinery and secretory functions are 
not altered; thus, the cell continues to secrete molecularly ho- 
mogeneous antibody. This antibody is indistinguishable 
from normal antibody molecules but is called myeloma pro- 
tein to denote its source. In a patient afflicted with multiple 
myeloma, myeloma protein can account for 95% of the 
serum immunoglobulins. In most patients, the myeloma 
cells also secrete excessive amounts of light chains. These ex- 
cess light chains were first discovered in the urine of 
myeloma patients and were named Bence-Jones proteins, 
for their discoverer. 

Multiple myeloma also occurs in other animals. In mice it 
can arise spontaneously, as it does in humans, or conditions fa- 
voring myeloma induction can be created by injecting mineral 
oil into the peritoneal cavity. The clones of malignant plasma 
cells that develop are called plasmacytomas, and many of these 
are designated MOPCs, denoting the mineral-oil induction of 
plasmacytoma cells. A large number of mouse MOPC lines se- 
creting different immunoglobulin classes are presently carried 
by the American Type-Culture Collection, a nonprofit reposi- 
tory of cell lines commonly used in research. 

Light-Chain Sequencing Revealed That 
Immunoglobulins Have Constant and 
Variable Regions 

When the amino acid sequences of several Bence-Jones pro- 
teins (light chains) from different individuals were com- 
pared, a striking pattern emerged. The amino-terminal half 
of the chain, consisting of 100-110 amino acids, was found 
to vary among different Bence-Jones proteins. This region 



was called the variable (V) region. The carboxyl-terminal 
half of the molecule, called the constant (C) region, had two 
basic amino acid sequences. This led to the recognition that 
there were two light chain types, kappa (k) and lambda (\). 
In humans, 60% of the light chains are kappa and 40% are 
lambda, whereas in mice, 95% of the light chains are kappa 
and only 5% are lambda. A single antibody molecule con- 
tains only one light chain type, either k or X, never both. 

The amino acid sequences of X light chains show minor dif- 
ferences that are used to classify X light chains into subtypes. In 
mice, there are three subtypes (XI, X2, and X3); in humans, 
there are four subtypes. Amino acid substitutions at only a few 
positions are responsible for the subtype differences. 

Heavy-Chain Sequencing Revealed Five Basic 
Varieties of Heavy Chains 

For heavy-chain sequencing studies, myeloma proteins were 
reduced with mercaptoethanol and alkylated, and the heavy 
chains were separated by gel filtration in a denaturing sol- 
vent. When the amino acid sequences of several myeloma 
protein heavy chains were compared, a pattern similar to that 
of the light chains emerged. The amino-terminal part of the 
chain, consisting of 100-110 amino acids, showed great se- 
quence variation among myeloma heavy chains and was 
therefore called the variable (V) region. The remaining part 
of the protein revealed five basic sequence patterns, corre- 
sponding to five different heavy-chain constant (C) regions 
(|x, 8, 7, e and a). Each of these five different heavy chains is 
called an isotype. The length of the constant regions is ap- 
proximately 330 amino acids for 8, 7, and a, and 440 amino 
acids for |x and e. The heavy chains of a given antibody mol- 
ecule determine the class of that antibody: IgM(|x), IgG(7), 
IgA(ct), IgD(8), or IgE(e). Each class can have either k or X 
light chains. A single antibody molecule has two identical 
heavy chains and two identical light chains, H 2 L 2 , or a multi- 
ple (H 2 L 2 )„ of this basic four-chain structure (Table 4-1). 

Minor differences in the amino acid sequences of the a 
and 7 heavy chains led to further classification of the heavy 
chains into subisotypes that determine the subclass of anti- 
body molecules they constitute. In humans, there are two 
subisotypes of a heavy chains — al and ct2 — (and thus two 
subclasses, IgAl and IgA2) — and four subisotypes of 7 heavy 
chains: 7I, 72, 73, and 74 (therefore four subclasses, IgGl, 
IgG2, IgG3, and IgG4). In mice, there are four subisotypes, 
7I, 72a, 72b, and 73, and the corresponding subclasses. 



Immunoglobulin Fine Structure 

The structure of the immunoglobulin molecule is deter- 
mined by the primary, secondary, tertiary, and quaternary 
organization of the protein. The primary structure, the 
amino acid sequence, accounts for the variable and constant 
regions of the heavy and light chains. The secondary struc- 
ture is formed by folding of the extended polypeptide chain 



Co to www.whfreeman.t 



lology ^^f Molecular Visualization 



>m/irr 

inoglobulin Structure 



> 



8536d_ch04_076-104 9/5/02 6:19 AM Page 80 mac76 n 



Generation of B-Cell and T-Cell Respor 



•yl, 72, ~/3, ~y4 


Kor\ 


72K2 
72^2 


None 


K ° 


(^K 2 )„ 
(^2)„ 


«1,a2 


Kor\ 


(a 2 K 2 )„ 

(<* 2 x 2 )„ 



back and forth upon itself into an antiparallel (3 pleated sheet 
(Figure 4-4). The chains are then folded into a tertiary struc- 
ture of compact globular domains, which are connected to 
neighboring domains by continuations of the polypeptide 
chain that lie outside the (3 pleated sheets. Finally, the globu- 
lar domains of adjacent heavy and light polypeptide chains 
interact in the quaternary structure (Figure 4-5), forming 
functional domains that enable the molecule to specifically 
bind antigen and, at the same time, perform a number of bi- 
ological effector functions. 

Immunoglobulins Possess Multiple Domains 
Based on the Immunoglobulin Fold 

Careful analysis of the amino acid sequences of immunoglob- 
ulin heavy and light chains showed that both chains contain 



several homologous units of about 110 amino acid residues. 
Within each unit, termed a domain, an intrachain disulfide 
bond forms a loop of about 60 amino acids. Light chains con- 
tain one variable domain (V L ), and one constant domain 
(C L ); heavy chains contain one variable domain ( V H ), and ei- 
ther three or four constant domains (C H 1, C H 2, C H 3, and 
C H 4), depending on the antibody class (Figure 4-6). 

X-ray crystallographic analysis revealed that im- 
munoglobulin domains are folded into a characteristic com- 
pact structure called the immunoglobulin fold. This 
structure consists of a "sandwich" of two (3 pleated sheets, 
each containing antiparallel (3 strands of amino acids, which 
are connected by loops of various lengths (Figure 4-7). The (3 
strands within a sheet are stabilized by hydrogen bonds that 
connect the -NH groups in one strand with carbonyl groups 
of an adjacent strand (see Figure 4-4). The (3 strands are 
characterized by alternating hydrophobic and hydrophilic 
amino acids whose side chains are arranged perpendicular to 
the plane of the sheet; the hydrophobic amino acids are ori- 
ented toward the interior of the sandwich, and the hy- 
drophilic amino acids face outward. 

The two (3 sheets within an immunoglobulin fold are sta- 
bilized by the hydrophobic interactions between them and by 
the conserved disulfide bond. An analogy has been made to 
two pieces of bread, the butter between them, and a tooth- 
pick holding the slices together. The bread slices represent the 
two (3 pleated sheets; the butter represents the hydrophobic 
interactions between them; and the toothpick represents the 
intrachain disulfide bond. Although variable and constant 
domains have a similar structure, there are subtle differences 
between them. The V domain is slightly longer than the C do- 
main and contains an extra pair of (3 strands within the |3- 
sheet structure, as well as the extra loop sequence connecting 
this pair of (3 strands (see Figure 4-7). 

The basic structure of the immunoglobulin fold con- 
tributes to the quaternary structure of immunoglobulins 
by facilitating noncovalent interactions between domains 



H — s 




I Structural formula of a (3 pleated sheet containing two 
antiparallel B strands. The structure is held together by hydrogen 
bonds between peptide bonds of neighboring stretches of polypep- 
tide chains. The amino acid side groups (R) are arranged perpendic- 



ular to the plane of the sheet. [Adapted from H. Lodish et al., 1995, 
Molecular Cell Biology, 4th ed., Scientific American Books, New York; 
reprinted by permission ofW. H. Freeman and Company.] 



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9/6/02 9:02 PM Page 81 mac85 Mac 85:365_E 




] Ribbon representation of an intact monoclonal ar 

body depicting the heavy chains (yellow and blue) and light chai 
(red). The domains of the molecule composed of (3 pleated shee 
are readily visible as is the extended conformation of the hinge 



gion. [The laboratory of A. McPherson provided this image, which is 
based on x-ray crystallography data determined by L.J. Harris et al., 
1992, Nature 360:369. The image was generated using the computer 
program RIBBONS.] 



across the faces of the (3 sheets (Figure 4-8). Interactions 
form links between identical domains (e.g., C H 2/C H 2, 
C H 3/C H 3, and C H 4/C H 4) and between nonidentical do- 
mains (e.g., V H /V L and C H 1/C L ). The structure of the im- 
munoglobulin fold also allows for variable lengths and 



sequences of amino acids that form the loops connecting 
the P strands. As the next section explains, some of the 
loop sequences of the V H and V L domains contain variable 
amino acids and constitute the antigen-binding site of the 
molecule. 




activity 




* (a) Heavy and light chains 
each containing about 110 amino acid re: 
disulfide bond that forms a loop of 60 a 
terminal domains, corresponding to the V 



effector func 



nediated by the other dor 



n that replaces the hin 



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Generation of B-Cell and T-Cell Respor 







| (a) Diagram of an immunoglobulin light chain depict- 
ing the immunoglobulin-fold structure of its variable and constant 
domains. The two B. pleated sheets in each domain are held together 
by hydrophobic interactions and the conserved disulfide bond. The (3 
strands that compose each sheet are shown in different colors. The 
amino acid sequences in three loops of each variable domain show 
considerable variation; these hypervariable regions (blue) make up 
the antigen-binding site. Hypervariable regions are usually called 



CDRs (complementarity-determining regions). Heavy-chain do- 
mains have the same characteristic structure, (b) The f3 pleated 
sheets are opened out to reveal the relationship of the individual (3 
strands and joining loops. Note that the variable domain contains 
two more B strands than the constant domain. [Part (a) adapted 
from M. Schiffer et al., 1973, Biochemistry 12:462o; reprinted with 
permission; part (b) adapted from Williams and Barclay, 1988, Annu. 
Rev. Immunol. 6:381.] 



Diversity in the Variable-Region Domain 
Is Concentrated in CDRs 

Detailed comparisons of the amino acid sequences of a large 
number of V L and V H domains revealed that the sequence 
variation is concentrated in a few discrete regions of these 
domains. The pattern of this variation is best summarized by 
a quantitative plot of the variability at each position of the 
polypeptide chain. The variability is defined as: 



Variability = 



# of different amino acids at a given position 



Frequency of the most common amino acid 
at given position 



Thus if a comparison of the sequences of 100 heavy chains 
revealed that a serine was found in position 7 in 51 of the se- 
quences (frequency 0.51), it would be the most common 
amino acid. If examination of the other 49 sequences showed 
that position 7 was occupied by either glutamine, histidine, 
proline, or tryptophan, the variability at that position would 
be 9.8 (5/0.51). Variability plots ofV L and V H domains of hu- 
man antibodies show that maximum variation is seen in 
those portions of the sequence that correspond to the loops 
that join the (3 strands (Figure 4-9). These regions were orig- 
inally called hypervariable regions in recognition of their 
high variability. Hypervariable regions form the antigen- 
binding site of the antibody molecule. Because the antigen 
binding site is complementary to the structure of the epitope, 



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Antibodies: Structure and Function chaf 



C L domain q \ 



Antigen-binding si 
V L domain 




22 


B0 Interact! 


)ns between domains in the separate chains 
molecule are critical to its quaternary struc- 


re. (a 

alysi 


) Model of IgC 
, showing assoc 
an amino acid r 
3 light chains a 


molecule, based on x-ray crystallography 
ations between domains. Each solid ball rep- 
;sidue; the larger tan balls are carbohydrate, 
e shown in shades of red; the two heavy 




in shades of blu 


e. (b) A schematic diagram showing the in- 



teracting heavy- and light-chain domains. Note that the C H 2/C H 2 
domains protrude because of the presence of carbohydrate (tan) in 
the interior. The protrusion makes this domain more accessible, en- 
abling it to interact with molecules such as certain complement 
components. [Part (a) from E. W. Silverton et al., 1977, Proc. Nat. 
Acad. Sci. U.S.A. 74:5140.] 



these areas are now more widely called complementarity de- 
termining regions (CDRs). The three heavy-chain and three 
light-chain CDR regions are located on the loops that con- 
nect the (3 strands of the V H and V L domains. The remainder 
of the V L and V H domains exhibit far less variation; these 
stretches are called the framework regions (FRs). The wide 
range of specificities exhibited by antibodies is due to varia- 
tions in the length and amino acid sequence of the six CDRs 
in each Fab fragment. The framework region acts as a scaf- 
fold that supports these six loops. The three-dimensional 
structure of the framework regions of virtually all antibodies 



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analyzed to date can be superimposed on one another; in 
contrast, the hypervariable loops (i.e., the CDRs) have differ- 
ent orientations in different antibodies. 



CDRs Bind Antigen 

The finding that CDRs are the antigen-binding regions of 
antibodies has been confirmed directly by high-resolution 
x-ray crystallography of antigen-antibody complexes. Crys- 
tallographic analysis has been completed for many Fab 
fragments of monoclonal antibodies complexed either with 



o to www.whfreeman.ee 
itibody Recognition of. 



nology 



^A Molecular Visualization 



9/6/02 9:02 PM Page 84 mac85 Mac 85:365_E 



Generation of B-Cell and T-Cell Respor 



CDR1 CDR2 




Residue position number 



Variability of ar 



s of hi 



bodies with different specificities. Thn 
(HV) regions, also called complementarity-det 
(CDRs), are present in both heavy- and light-cha 

Figure 4-7 (right), the three HV regio 



Residue position number 

light-chain V domain are brought into proximity in the folded struc- 
ture. The same is true of the heavy-chain V domain. [Based on E. A. 
Kabat et al., 1977, Sequence of Immunoglobulin Chains, U.S. Dept. 
of Health, Education, and Welfare.] 



;. As 



large globular protein antigens or with a number of smaller 
antigens including carbohydrates, nucleic acids, peptides, 
and small haptens. In addition, complete structures have 
been obtained for several intact monoclonal antibodies. X- 
ray diffraction analysis of antibody-antigen complexes has 



shown that several CDRs may make contact with the antigen, 
and a number of complexes have been observed in which all 
six CDRs contact the antigen. In general, more residues in the 
heavy-chain CDRs appear to contact antigen than in the 
light-chain CDRs. Thus the V H domain often contributes 




I*' (a) Side view of the three-dimensional structure of 
the combining site of an angiotensin II — Fab complex. The peptide is 
in red. The three heavy-chain CDRs (HI, H2, H3) and three light- 
chain CDRs (LI, L2, L3) are each shown in a different color. All six 
CDRs contain side chains, shown in yellow, that are within van der 



r Waals 


tact of the angiote 


be 


n peptide, 
tween an^ 


(b) 


Side viev 


of the v 
d Fab fra 


s- 


»nt. [Fr 


m K. C. Cara 
Johns Hopkins 


Un 


al. 


1992, Sci 
sity.j 




257:502 


courtesy 


of 



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8536d_ch04_076-104 9/5/02 6:19 AM Page 85 mac76 n 



more to antigen binding than the V L domain. The dominant 
role of the heavy chain in antigen binding was demonstrated 
in a study in which a single heavy chain specific for a glyco- 
protein antigen of the human immunodeficiency virus 
(HIV) was combined with various light chains of different 
antigenic specificity. All of the hybrid antibodies bound to 
the HIV glycoprotein antigen, indicating that the heavy chain 
alone was sufficient to confer specificity. However, one 
should not conclude that the light chain is largely irrelevant; 
in some antibody-antigen reactions, the light chain makes 
the more important contribution. 

The actual shape of the antigen binding site formed by 
whatever combination of CDRs are used in a particular anti- 
body has been shown to vary dramatically. As pointed out in 
Chapter 3, contacts between a large globular protein antigen 
and antibody occur over a broad, often rather flat, undulat- 
ing face. In the area of contact, protrusions or depressions on 
the antigen are likely to match complementary depressions 
or protrusions on the antibody. In the case of the well studied 
lysozyme/anti-lysozyme system, crystallographic studies 
have shown that the surface areas of interaction are quite 
large, ranging from about 650 A 2 to more than 900 A 2 . 
Within this area, some 15-22 amino acids in the antibody 
contact the same number of residues in the protein antigen. 
In contrast, antibodies bind smaller antigens, such as small 
haptens, in smaller, recessed pockets in which the ligand is 
buried. This is nicely illustrated by the interaction of the 



small octapeptide hormone angiotensin II with the binding 
site of an anti-angiotensin antibody (Figure 4-10). 

Conformational Changes May Be 
Induced by Antigen Binding 

As more x-ray crystallographic analyses of Fab fragments 
were completed, it became clear that in some cases binding of 
antigen induces conformational changes in the antibody, 
antigen, or both. Formation of the complex between neur- 
aminidase and anti-neuraminidase is accompanied by a 
change in the orientation of side chains of both the epitope 
and the antigen-binding site. This conformational change re- 
sults in a closer fit between the epitope and the antibody's 
binding site. 

In another example, comparison of an anti-hemagglutinin 
Fab fragment before and after binding to a hemagglutinin 
peptide antigen has revealed a visible conformational change 
in the heavy-chain CDR3 loop and in the accessible surface of 
the binding site. Another striking example of conformational 
change has been seen in the complex between an Fab frag- 
ment derived from a monoclonal antibody against the HIV 
protease and the peptide epitope of the protease. As shown in 
Figure 4-11, there are significant changes in the Fab upon 
binding. In fact, upon antigen binding, the CDR1 region of 
the light chain moves as much as 1 A and the heavy chain 
CDR3 moves 2.7 A. Thus, in addition to variability in the 




from HIV protease and an Fab fragment from an anti-protease anti- 
body (left) and comparison of the Fab structure before and after pep- 
tide binding [right). In the right panel, the red line shows the 
structure ofthe Fab fragment before it binds the peptide and the blue 



ine shows its structure when bound. There are significant confoi 
mational changes in the CDRs ofthe Fab on binding the antiger 
These are especially pronounced in the light chain CDR1 (LI) am 
the heavy chain CDR3 (H3). [From J. Lescar et al., 1997, j. Mol. Bio 
267:1207; courtesy ofC. Bentley, Institute Pasteur] 



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Generation of B-Cell and T-Cell Respor 



length and amino acid composition of the CDR loops, the 
ability of these loops to significantly change conformation 
upon antigen binding enables antibodies to assume a shape 
more effectively complementary to that of their epitopes. 

As already indicated, conformational changes following 
antigen binding need not be limited to the antibody. Al- 
though it is not shown in Figure 4-11, the conformation of 
the protease peptide bound to the Fab shows no structural 
similarity to the corresponding epitope in the native HIV 
protease. It has been suggested that the inhibition of protease 
activity by this anti-HIV protease antibody is a result of its 
distortion of the enzyme's native confor 



Constant-Region Domains 

The immunoglobulin constant-region 
; biological functions that are 
acid sequence of each domain. 



take part in 
determined by the 



C H i AND C l DOMAINS 

The C H 1 and C L domains serve to extend the Fab arms of the 
antibody molecule, thereby facilitating interaction with anti- 
gen and increasing the maximum rotation of the Fab arms. 
In addition, these constant-region domains help to hold the 
V H and V L domains together by virtue of the interchain 
disulfide bond between them (see Figure 4-6). Also, the C H 1 
and C L domains may contribute to antibody diversity by al- 
lowing more random associations between V H and V L do- 
mains than would occur if this association were driven by the 



V H /V L interaction alone. These considerations have impor- 
tant implications for building a diverse repertoire of anti- 
bodies. As Chapter 5 will show, random rearrangements of 
the immunoglobulin genes generate unique V H and V L se- 
quences for the heavy and light chains expressed by each B 
lymphocyte; association of the V H and V L sequences then 
generates a unique antigen-binding site. The presence of C H 1 
and C L domains appears to increase the number of stable V H 
and V L interactions that are possible, thus contributing to the 
overall diversity of antibody molecules that can be expressed 
by an animal. 

HINGE REGION 

The 7, 8, and a heavy chains contain an extended peptide se- 
quence between the C H 1 and C H 2 domains that has no ho- 
mology with the other domains (see Figure 4-8). This region, 
called the hinge region, is rich in proline residues and is flex- 
ible, giving IgG, IgD, and IgA segmental flexibility. As a result, 
the two Fab arms can assume various angles to each other 
when antigen is bound. This flexibility of the hinge region 
can be visualized in electron micrographs of antigen-anti- 
body complexes. For example, when a molecule containing 
two dinitrophenol (DNP) groups reacts with anti-DNP anti- 
body and the complex is captured on a grid, negatively 
stained, and observed by electron microscopy, large com- 
plexes (e.g., dimers, trimers, tetramers) are seen. The angle 
between the arms of the Y-shaped antibody molecules differs 
in the different complexes, reflecting the flexibility of the 
hinge region (Figure 4-12). 







Ag-Ab Trimer 



hinge region in antibody molecules, (a) A hapten in which two dini- 
trophenyl (DNP) groups are tethered by a short connecting spacer 
group reacts with anti-DNP antibodies to form trimers, tetramers, 
and other larger antigen-antibody complexes. A trimer is shown 
schematically, (b) In an electron micrograph of a negatively stained 
preparation of these complexes, two triangular trimeric structures 



are clearly visible. The antibody protein stands out as a light struc- 
ture against the electron-dense background. Because ofthe flexibility 
of the hinge region, the angle between the arms ofthe antibody mol- 
ecules varies. [Photograph from R. C. Valentine and N. M. Green, 
1967, J. Mol. Biol. 27:615; reprinted by permission of Academic Press 
Inc. (London) Ltd.] 



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Antibodies: Structure and Functioi 



Two prominent amino acids in the hinge region are pro- 
line and cysteine. The large number of proline residues in the 
hinge region gives it an extended polypeptide conformation, 
making it particularly vulnerable to cleavage by proteolytic 
enzymes; it is this region that is cleaved with papain or pepsin 
(see Figure 4-3). The cysteine residues form interchain disul- 
fide bonds that hold the two heavy chains together. The num- 
ber of interchain disulfide bonds in the hinge region varies 
considerably among different classes of antibodies and be- 
tween species. Although (jl and e chains lack a hinge region, 
they have an additional domain of 110 amino acids 
(C H 2/C H 2) that has hingelike features. 

OTHER CONSTANT-REGION DOMAINS 
As noted already, the heavy chains in IgA, IgD, and IgG con- 
tain three constant-region domains and a hinge region, 
whereas the heavy chains in IgE and IgM contain four con- 
stant-region domains and no hinge region. The correspond- 
ing domains of the two groups are as follows: 



IgA, IgD, IgG 


IgE, IgM 


ChI/ChI 


C H 1/C H 1 


Hinge region 


C H 2/C H 2 


C H 2/C H 2 


C H 3/C H 3 


C H 3/C H 3 


C H 4/C H 4 



Although the C H 2/C H 2 domains in IgE and IgM occupy the 
same position in the polypeptide chains as the hinge region 
in the other classes of immunoglobulin, a function for this 
extra domain has not yet been determined. 

X-ray crystallographic analyses have revealed that the 
two C H 2 domains of IgA, IgD, and IgG (and the C H 3 do- 
mains of IgE and IgM) are separated by oligosaccharide side 
chains; as a result, these two globular domains are much 
more accessible than the others to the aqueous environ- 
ment (see Figure 4-8b). This accessibility is one of the ele- 
ments that contributes to the biological activity of these 
domains in the activation of complement components by 
IgG and IgM. 

The carboxyl-terminal domain is designated C H 3/ C H 3 in 
IgA, IgD, and IgG and C H 4/C H 4 in IgE and IgM. The five 
classes of antibody and their subclasses can be expressed ei- 
ther as secreted immunoglobulin (slg) or as membrane- 
bound immunoglobulin (mlg). The carboxyl-terminal 
domain in secreted immunoglobulin differs in both struc- 
ture and function from the corresponding domain in mem- 
brane-bound immunoglobulin. Secreted immunoglobulin 
has a hydrophilic amino acid sequence of various lengths at 
the carboxyl-terminal end. The functions of this domain in 
the various classes of secreted antibody will be described 
later. In membrane-bound immunoglobulin, the carboxyl- 
terminal domain c 



■ An extracellular hydrophilic "spacer" sequence 
composed of 26 amino acid residues 

■ A hydrophobic transmembrane sequence 

■ A short cytoplasmic tail 

The length of the transmembrane sequence is constant among 
all immunoglobulin isotypes, whereas the lengths of the extra- 
cellular spacer sequence and the cytoplasmic tail vary. 

B cells express different classes of mlg at different devel- 
opmental stages. The immature B cell, called a pre-B cell, ex- 
presses only mlgM; later in maturation, mlgD appears and is 
coexpressed with IgM on the surface of mature B cells before 
they have been activated by antigen. A memory B cell can ex- 
press mlgM, mlgG, mlgA, or mlgE. Even when different 
classes are expressed sequentially on a single cell, the anti- 
genic specificity of all the membrane antibody molecules ex- 
pressed by a single cell is identical, so that each antibody 
molecule binds to the same epitope. The genetic mechanism 
that allows a single B cell to express multiple immunoglobu- 
lin isotypes all with the same antigenic specificity is de- 
scribed in Chapter 5. 



Antibody- Mediated 
Effector Functions 

In addition to binding antigen, antibodies participate in a 
broad range of other biological activities. When considering 
the role of antibody in defending against disease, it is impor- 
tant to remember that antibodies generally do not kill or 
remove pathogens solely by binding to them. In order to 
be effective against pathogens, antibodies must not only 
recognize antigen, but also invoke responses — effector 
functions — that will result in removal of the antigen and 
death of the pathogen. While variable regions of antibody are 
the sole agents of binding to antigen, the heavy-chain con- 
stant region (C H ) is responsible for a variety of collaborative 
interactions with other proteins, cells, and tissues that result 
in the effector functions of the humoral response. 

Because these effector functions result from interactions 
between heavy-chain constant regions and other serum pro- 
teins or cell-membrane receptors, not all classes of im- 
munoglobulin have the same functional properties. An 
overview of four major effector functions mediated by do- 
mains of the constant region is presented here. A fifth func- 
tion unique to IgE, the activation of mast cells, eosinophils, 
and basophils, will be described later. 

Opsonization Is Promoted by Antibody 

Opsonization, the promotion of phagocytosis of antigens by 
macrophages and neutrophils, is an important factor in an- 
tibacterial defenses. Protein molecules called Fc receptors 
(FcR), which can bind the constant region of Ig molecules, 
are present on the surfaces of macrophages and neutrophils. 



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Generation of B-Cell and T-Cell Respor 



The binding of phagocyte Fc receptors with several antibody 
molecules complexed with the same target, such as a bacter- 
ial cell, produces an interaction that results in the binding of 
the pathogen to the phagocyte membrane. This crosslinking 
of the FcR by binding to an array of antibody Fc regions ini- 
tiates a signal-transduction pathway that results in the 
phagocytosis of the antigen-antibody complex. Inside the 
phagocyte, the pathogen becomes the target of various de- 
structive processes that include enzymatic digestion, oxida- 
tive damage, and the membrane-disrupting effects of 
antibacterial peptides. 

Antibodies Activate Complement 

IgM and, in humans, most IgG subclasses can activate a col- 
lection of serum glycoproteins called the complement sys- 
tem. Complement includes a collection of proteins that can 
perforate cell membranes. An important byproduct of the 
complement activation pathway is a protein fragment called 
C3b, which binds nonspecifically to cell- and antigen-anti- 
body complexes near the site at which complement was acti- 
vated. Many cell types — for example, red blood cells and 
macrophages — have receptors for C3b and so bind cells or 
complexes to which C3b has adhered. Binding of adherent 
C3b by macrophages leads to phagocytosis of the cells or 
molecular complexes attached to C3b. Binding of antigen- 
antibody complexes by the C3b receptors of a red blood cell 
allows the erythrocyte to deliver the complexes to liver or 
spleen, where resident macrophages remove them without 
destroying the red cell. The collaboration between antibody 
and the complement system is important for the inactivation 
and removal of antigens and the killing of pathogens. The 
process of complement activation is described in detail in 
Chapter 13. 

Antibody-Dependent Cell- Mediated 
Cytotoxicity (ADCC) Kills Cells 

The linking of antibody bound to target cells (virus infected 
cells of the host) with the Fc receptors of a number of cell 
types, particularly natural killer (NK) cells, can direct the cy- 
totoxic activities of the effector cell against the target cell. In 
this process, called antibody-dependent cell-mediated cyto- 
toxicity (ADCC), the antibody acts as a newly acquired re- 
ceptor enabling the attacking cell to recognize and kill 
the target cell. The phenomenon of ADCC is discussed in 
Chapter 14. 

Some Antibodies Can Cross Epithelial 
Layers by Transcytosis 

The delivery of antibody to the mucosal surfaces of the respi- 
ratory, gastrointestinal, and urogenital tracts, as well as its ex- 
port to breast milk, requires the movement of immunoglob- 
ulin across epithelial layers, a process called transcytosis. 
The capacity to be transported depends on properties of the 



constant region. In humans and mice, IgA is the major anti- 
body species that undergoes such transcytosis, although IgM 
can also be transported to mucosal surfaces. Some mam- 
malian species, such as humans and mice, also transfer sig- 
nificant amounts of most subclasses of IgG from mother to 
fetus. Since maternal and fetal circulatory systems are sepa- 
rate, antibody must be transported across the placental tissue 
that separates mother and fetus. In humans, this transfer 
takes place during the third trimester of gestation. The im- 
portant consequence is that the developing fetus receives a 
sample of the mother's repertoire of antibody as a protective 
endowment against pathogens. As with the other effector 
functions described here, the capacity to undergo transpla- 
cental transport depends upon properties of the constant re- 
gion of the antibody molecule. 

The transfer of IgG from mother to fetus is a form of pas- 
sive immunization, which is the acquisition of immunity by 
receipt of preformed antibodies rather than by active pro- 
duction of antibodies after exposure to antigen. The ability to 
transfer immunity from one individual to another by the 
transfer of antibodies is the basis of passive antibody therapy, 
an important and widely practiced medical procedure (see 
Clinical Focus). 



Antibody Classes and 
Biological Activities 



The various immunoglobulin isotypes and classes have 
been mentioned briefly already. Each class is distinguished 
by unique amino acid sequences in the heavy-chain con- 
stant region that confer class-specific structural and func- 
tional properties. In this section, the structure and effector 
functions of each class are described in more detail. The 
molecular properties and biological activities of the 
immunoglobulin classes are summarized in Table 4-2 
(page 90). The structures of the five major classes are dia- 
gramed in Figure 4-13 (page 91). 

Immunoglobulin G (IgG) 

IgG, the most abundant class in serum, constitutes about 
80% of the total serum immunoglobulin. The IgG molecule 
consists of two 7 heavy chains and two k or two X light chains 
(see Figure 4- 13a). There are four human IgG subclasses, dis- 
tinguished by differences in 7 -chain sequence and numbered 
according to their decreasing average serum concentrations: 
IgGl, IgG2, IgG3, and IgG4 (see Table 4-2). 

The amino acid sequences that distinguish the four IgG 
subclasses are encoded by different germ-line C H genes, 
whose DNA sequences are 90%-95% homologous. The 
structural characteristics that distinguish these subclasses 
from one another are the size of the hinge region and the 
number and position of the interchain disulfide bonds be- 
tween the heavy chains (Figure 4-14, page 92). The subtle 



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CLINICAL FOCUS 



Passive Antibody Therapy 



EmilBehringand 
Shibasaburo Kitasato reported an extra- 
ordinary experiment. They immunized 
rabbits with tetanus and then collected 
serum from these animals. Subse- 
quently, they injected 0.2 ml of the im- 
mune serum into the abdominal cavity of 
six mice. After 24 hours, they infected the 
treated animals and untreated controls 
with live, virulent tetanus bacteria. All of 
the control mice died within 48 hours of 
infection, whereas the treated mice not 
only survived but showed no effects of 
infection. This landmark experiment 
demonstrated two important points. 
One, it showed that following immuniza- 
tion, substances appeared in serum that 
could protect an animal against path- 
ogens. Two, this work demonstrated that 
immunity could be passively acquired. 
Immunity could be transferred from one 
animal to another by taking serum from 
an immune animal and injecting it into a 
nonimmune one. These and subsequent 
experiments did not go unnoticed. Both 
men eventually received titles (Behring 
became von Behring and Kitasato be- 
came Baron Kitasato). A few years later, 
in 1901, von Behring was awarded the 
first Nobel prize in Medicine. 

These early observations and others 
paved the way for the introduction of 
passive immunization into clinical prac- 



tice. During the 1930s and 1940s, pas- 
sive immunotherapy, the endowment of 
resistance to pathogens by transfer of 
the agent of immunity from an immu- 
nized donor to an unimmunized recipi- 
ent, was used to prevent or modify the 
course of measles and hepatitis A. Dur- 
ing subsequent years, clinical experience 
and advances in the technology of prepa- 
ration of immunoglobulin for passive 
immunization have made this approach 
a standard medical practice. Passive im- 
munization based on the transfer of anti- 
bodies is widely used in the treatment of 
immunodeficiency diseases and as a 
protection against anticipated exposure 
to infectious agents against which one 

Immunoglobulin for passive immu- 
nization is prepared from the pooled 
plasma of thousands of donors. In effect, 
recipients of these antibody preparations 
are receiving a sample of the antibodies 
produced by many people to a broad di- 
versity of different pathogens. In fact a 
gram of intravenous immune globulin 
(IVIC) contains about 10 18 molecules of 
antibody (mostly IgC) and may incorpo- 
rate more than 10 7 different antibody 
specificities. During the course of ther- 
apy, patients receive significant amounts 
of IVIC, usually 200-400 mg per kilo- 
gram of body weight. This means that 
an immunodeficient patient weighing 



70 kilograms would receive 14 to 28 
grams of IVIC every 3 to 4 weeks. A prod- 
uct derived from the blood of such a 
large number of donors carries a risk of 
harboring pathogenic agents, particu- 
larly viruses. The risk is minimized by 
the processes used to produce intra- 



i. The n 



lufac- 



ture of IVIG involves treatment with 
solvents, such as ethanol, and the use of 
detergents that are highly effective in 
inactivating viruses such as HIV and he- 
patitis. In addition to removing or inacti- 
vating infectious agents, the production 
process must also eliminate aggregated 
immunoglobulin, because antibody ag- 
gregates can trigger massive activation 
of the complement pathway, leading to 
severe, even fatal, anaphylaxis. 

Passively administered antibody ex- 
erts its protective action in a number of 
ways. One of the most important is the 
recruitment of the complement pathway 
to the destruction or removal of a 
pathogen. In bacterial infections, anti- 
body binding to bacterial surfaces pro- 
motes opsonization, the phagocytosis 
and killing of the invader by macro- 
phages and neutrophils. Toxins and 
viruses can be bound and neutralized by 
antibody, even as the antibody marks the 
pathogen for removal from the body by 
phagocytes and by organs such as liver 
and kidneys. By the initiation of antibody- 
dependent cell-mediated cytotoxicity 
(ADCC), antibodies can also mediate the 
killing of target cells by cytotoxic cell pop- 
ulations such as natural killer cells. 



amino acid differences between subclasses of IgG affect the 
biological activity of the molecule: 

■ IgGl, IgG3, and IgG4 readily cross the placenta and play 
an important role in protecting the developing fetus. 

■ IgG3 is the most effective complement activator, 
followed by IgGl; IgG2 is less efficient, and IgG4 is not 
able to activate complement at all. 

■ IgGl and IgG3 bind with high affinity to Fc receptors on 
phagocytic cells and thus mediate opsonization. IgG4 
has an intermediate affinity for Fc receptors, and IgG2 
has an extremely low affinity. 



Immunoglobulin M (IgM) 

IgM accounts for 5%-10% of the total serum immunoglob- 
ulin, with an average serum concentration of 1.5 mg/ml. 
Monomeric IgM, with a molecular weight of 180,000, is ex- 
pressed as membrane-bound antibody on B cells. IgM is se- 
creted by plasma cells as a pentamer in which five monomer 
units are held together by disulfide bonds that link their car- 
boxyl-terminal heavy chain domains (C (JL 4/C |X 4) and their 
0^3/0^3 domains (see Figure 4-13e). The five monomer 
subunits are arranged with their Fc regions in the center 
of the pentamer and the ten antigen-binding sites on the 
periphery of the molecule. Each pentamer contains an 



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Generation of B-Cell and T-Cell Respor 



| TABLE 4-2 [ 


Property/Activity 


lgC1 


lgC2 


lgC3 


lgC4 


IgAI 


lgA2 


IgM* 


"gE 


IgD 


Molecular weight 1 " 


150,000 


150,000 


150,000 


150,000 


150,000- 
600,000 


150,000- 
600,000 


900,000 


190,000 


150,000 


Heavy-chain 
component 


■yl 


y2 


73 


7 4 


«1 


a2 


, 


6 




Normal serum 
level (mg/ml) 


9 


3 


1 


0.5 


3.0 


0.5 


1.5 


0.0003 


0.03 


half life (days) 


23 


23 


8 


23 


6 


6 


5 


2.5 


3 


Activates classical 
complement 
pathway 


+ 


+ /" 


+ + 








+ + + 






Crosses placenta 


+ 


+ /" 


+ 


+ 


- 


- 


- 


- 


- 


Present on 
membrane of 


~ 


~~ 


~ 


~ 


~ 


~ 


+ 


~ 


+ 


Binds to Fc 
receptors of 
phagocytes 


+ + 


+ /- 


+ + 


+ 


" 


" 


? 


" 


" 


Mucosal transport 


" 


- 


" 


- 


+ + 


+ + 


+ 


" 


" 


degranulation 


" 


' 


" 


" 


" 


" 


" 


+ 


" 


"Activity levels indicated a 


s follows: ++ = 


high; + = m 


oderate; +/- = 


Animal; - = 




,onable. 








T|gG, IgE, and IgD always 
IgM is a monomer, but se 


creted IgM in s 


ers;lgAcane 


xirtasamc 


r.dimer.trim 


er.ortetramer.r. 


embrane-bound 








JlgM is the first isotype p 


oducedbythe 


neonate and d 




nmune respo 


nse. 











additional Fc-linked polypeptide called the J (joining) 
chain, which is disulfide-bonded to the carboxyl-terminal 
cysteine residue of two of the ten jjl chains. The J chain ap- 
pears to be required for polymerization of the monomers to 
form pentameric IgM; it is added just before secretion of the 
pentamer. 

IgM is the first immunoglobulin class produced in a pri- 
mary response to an antigen, and it is also the first im- 
munoglobulin to be synthesized by the neonate. Because of 
its pentameric structure with 10 antigen-binding sites, serum 
IgM has a higher valency than the other isotypes. An IgM 
molecule can bind 10 small hapten molecules; however, be- 
cause of steric hindrance, only 5 or fewer molecules of larger 
antigens can be bound simultaneously. Because of its high va- 
lency, pentameric IgM is more efficient than other isotypes in 
binding antigens with many repeating epitopes such as viral 
particles and red blood cells (RBCs). For example, when 
RBCs are incubated with specific antibody, they clump to- 
gether into large aggregates in a process called agglutination. 
It takes 100 to 1000 times more molecules of IgG than of IgM 
to achieve the same level of agglutination. A similar phenom- 
enon occurs with viral particles: less IgM than IgG is required 



to neutralize viral infectivity. IgM is also more efficient than 
IgG at activating complement. Complement activation re- 
quires two Fc regions in close proximity, and the pentameric 
structure of a single molecule of IgM fulfills this requirement. 
Because of its large size, IgM does not diffuse well and 
therefore is found in very low concentrations in the intercel- 
lular tissue fluids. The presence of the I chain allows IgM to 
bind to receptors on secretory cells, which transport it across 
epithelial linings to enter the external secretions that bathe 
mucosal surfaces. Although IgA is the major isotype found 
in these secretions, IgM plays an important accessory role as 
a secretory immunoglobulin. 

Immunoglobulin A (IgA) 

Although IgA constitutes only 10%- 15% of the total im- 
munoglobulin in serum, it is the predominant im- 
munoglobulin class in external secretions such as breast 
milk, saliva, tears, and mucus of the bronchial, genitouri- 
nary, and digestive tracts. In serum, IgA exists primarily as a 
monomer, but polymeric forms (dimers, trimers, and some 
tetramers) are sometimes seen, all containing a J-chain 



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Antibodies: Structure and Function chaf 



(d) IgA (dimer) 





' General structures of the five major classes of se- 
creted antibody. Light chains are shown in shades of pink, disulfide 
bonds are indicated by thick black lines. Note that the IgC, IgA, and 
IgD heavy chains (blue, orange, and green, respectively) contain four 
domains and a hinge region, whereas the IgM and IgE heavy chains 
(purple and yellow, respectively) contain five domains but no hinge 
region. The polymeric forms of IgM and IgA contain a polypeptide, 



called the j chain, that is linked by two disulfide bonds to the Fc 
gion in two different monomers. Serum IgM is always a pentam 

even tetramers are sometimes present. Not shown in these figur 
are intrachain disulfide bonds and disulfide bonds linking light ai 
heavy chains (see Figure 4-2). 



polypeptide (see Figure 4- 13d). The IgA of external s 
tions, called secretory IgA, consists of a dime 
J-chain polypeptide, and a polypeptide chain called secre- 
tory component (Figure 4-15a, page 93). As is explained be- 
low, secretory component is derived from the receptor that is 
responsible for transporting polymeric IgA across cell mem- 
branes. The J-chain polypeptide in IgA is identical to that 
found in pentameric IgM and serves a similar function in fa- 



cilitating the polymerization of both serum IgA and secre- 
tory IgA. The secretory component is a 70,000-MW polypep- 
tide produced by epithelial cells of mucous membranes. It 
consists of five immunoglobulin-like domains that bind to 
the Fc region domains of the IgA dimer. This interaction is 
stabilized by a disulfide bond between the fifth domain of the 
secretory component and one of the chains of the dimeric 
IgA. 



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Generation of B-Cell and T-Cell Respor 




eral structure of the four : 
IgC, which differ in the number and arrangerr 



jbclasses of hum 
:nt of the interch; 



disulfide bonds (thick black lii 
feature of human lgC3 is its 



;s) linking the heavy chains. A notable 
interchain disulfide bonds. 



The daily production of secretory IgA is greater than that 
loglobulin class. IgA-secreting plasma 
•ated along mucous membrane surfaces. 
Along the jejunum of the small intestine, for example, there 
are more than 2.5 X 10 10 IgA-secreting plasma cells — a 
number that surpasses the total plasma cell population of the 
bone marrow, lymph, and spleen combined! Every day, a hu- 
s from 5 g to 15 g of secretory IgA i 



The plasma cells that produce IgA preferentially migrate 
(home) to subepithelial tissue, where the secreted IgA binds 
tightly to a receptor for polymeric immunoglobulin mole- 
cules (Figure 4-15b). This poly-Ig receptor is expressed on 
the basolateral surface of most mucosal epithelia (e.g., the 
lining of the digestive, respiratory, and genital tracts) and on 
glandular epithelia in the mammary, salivary, and lacrimal 
glands. After polymeric IgA binds to the poly-Ig receptor, the 
receptor-IgA complex is transported across the epithelial 
barrier to the lumen. Transport of the receptor-IgA complex 
involves receptor-mediated endocytosis into coated pits and 
directed transport of the vesicle across the epithelial cell to 
the luminal membrane, where the vesicle fuses with the 
plasma membrane. The poly-Ig receptor is then cleaved en- 
zymatically from the membrane and becomes the secretory 
component, which is bound to and released together with 
polymeric IgA into the mucous secretions. The secretory 
component masks sites susceptible to protease cleavage in the 
hinge region of secretory IgA, allowing the polymeric mole- 
cule to exist longer in the protease-rich mucosal environ- 
ment than would be possible otherwise. Pentameric IgM is 
also transported into mucous secretions by this mechanism, 
although it accounts for a much lower percentage of anti- 
body in the mucous secretions than does IgA. The poly-Ig re- 
ceptor interacts with the J chain of both polymeric IgA and 
IgM antibodies. 

Secretory IgA serves an important effector function at 
nbrane surfaces, which are the main entry sites 



for most pathogenic organisms. Because it is polymeric, se- 
cretory IgA can cross-link large antigens with multiple epi- 
topes. Binding of secretory IgA to bacterial and viral surface 
antigens prevents attachment of the pathogens to the mu- 
cosal cells, thus inhibiting viral infection and bacterial colo- 
nization. Complexes of secretory IgA and antigen are easily 
entrapped in mucus and then eliminated by the ciliated ep- 
ithelial cells of the respiratory tract or by peristalsis of the 
gut. Secretory IgA has been shown to provide an important 
line of defense against bacteria such as Salmonella, Vibrio 
cholerae, and Neisseria gonorrhoeae and viruses such as polio, 
influenza, and reovirus. 

Breast milk contains secretory IgA and many other mole- 
cules that help protect the newborn against infection during 
the first month of life (Table 4-3). Because the immune sys- 
tem of infants is not fully functional, breast-feeding plays an 
important role in maintaining the health of newborns. 



Immunoglobulin E (IgE) 

The potent biological activity of IgE allowed it to be identi- 
fied in serum despite its extremely low average serum con- 
centration (0.3 jjug/ml). IgE antibodies mediate the immediate 
hypersensitivity reactions that are responsible for the symp- 
toms of hay fever, asthma, hives, and anaphylactic shock. The 
presence of a serum component responsible for allergic reac- 
tions was first demonstrated in 1921 by K. Prausnitz and 
H. Kustner, who injected serum from an allergic person 
intra-dermally into a nonallergic individual. When the 
appropriate antigen was later injected at the same site, a 
wheal and flare reaction (analogous to hives) developed 
there. This reaction, called the P-K reaction (named for its 
originators, Prausnitz and Kustner), was the basis for the first 
biological assay for IgE activity. 

Actual identification of IgE was accomplished by K. and 
T. Ishizaka in 1966. They obtained serum from an allergic in- 



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Antibodies: Structure 



(a) Structure of secretory IgA 




nation of secretory IgA 







' Structure and formation of secretory IgA. (a) Secre- 
tory IgA consists of at least two IgA molecules, which are covalently 
linked to each other through a j chain and are also covalently linked 
with the secretory component. The secretory component contains 
five Ig-like domains and is linked to dimeric IgA by a disulfide bond 
between its fifth domain and one of the IgA heavy chains, (b) Secre- 



tory IgA is formed during transport through mucous membrane 
epithelial cells. Dimeric IgA binds to a poly-lg receptor on the baso- 
lateral membrane of an epithelial cell and is internalized by receptor- 
mediated endocytosis. After transport of the receptor-lgA complex 
to the luminal surface, the poly-lg receptor is enzymatically cleaved, 
releasing the secretory component bound to the dimeric IgA. 



dividual and immunized rabbits with it to prepare anti- 
isotype antiserum. The rabbit antiserum was then allowed to 
react with each class of human antibody known at that time 
(i.e., IgG, IgA, IgM, and IgD). In this way, each of the known 
anti-isotype antibodies was precipitated and removed from 
the rabbit anti-serum. What remained was an anti-isotype 
antibody specific for an unidentified class of antibody. This 
antibody turned out to completely block the P-K reaction. 
The new antibody was called IgE (in reference to the E anti- 
gen of ragweed pollen, which is a potent inducer of this class 
of antibody). 



IgE binds to Fc receptors on the membranes of blood ba- 
sophils and tissue mast cells. Cross-linkage of receptor- 
bound IgE molecules by antigen (allergen) induces basophils 
and mast cells to translocate their granules to the plasma 
membrane and release their contents to the extracellular en- 
vironment, a process known as degranulation. As a result, a 
variety of pharmacologically active mediators are released 
and give rise to allergic manifestations (Figure 4-16). Local- 
ized mast-cell degranulation induced by IgE also may release 
mediators that facilitate a buildup of various cells necessary 
for antiparasitic defense (see Chapter 15). 



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Generation of B-Cell and T-Cell Respor 



Antibodies of 
secretory IgA class 

B 12 binding protein 

Bifidus factor 

Fatty acids 
Fibronectin 



growth factors 
Interferon (IFN--y) 
Lactoferrin 

Lysozyme 

Mucins 

Oligosaccharides 

SOURCE: Adapted from 



Bind to microbes in baby's digestive tract and thereby prevent their attachment to the walls of tl 
subsequent passage into the body's tissues. 

Reduces amount of vitamin B 12 , which bacteria need in order to grow. 

mless bacterium, in baby's gut. Growth of such rr 



Promotes growth of Lactobacillus bifidus 
bacteria helps to crowd out dangerous 
Disrupt membranes surrounding certaii 

antimicrobial activity of m 
le reactions in baby's gut. 



in viruses and destroy them, 
rophages; helps to repair tissu 



Binds to iron, a mineral many bacteria need tc 
thwarts growth of pathogenic bacteria. 



Kills bacteria by di< 

Adhere to bacteria 

Bindtomicroorgai 

lewman, 1995, How brea 



•upting their cell walls. 

ind viruses, thus keeping such microorganis 

isms and bar them from attaching to mucos 



s from attaching tc 






1. 273(6):76. 



laged by 

les lining the gut 




Histamine and 
other substances 
that mediate 



lergen cross-linkage of receptor-bound IgE c 
mast cells induces degranulation, causing release of substana 
(blue dots) that mediate allergic manifestations. 



Immunoglobulin D (IgD) 

IgD was first discovered when a patient developed a multiple 
myeloma whose myeloma protein failed to react with anti- 
isotype antisera against the then-known isotypes: IgA, IgM, 
and IgG. When rabbits were immunized with this myeloma 
protein, the resulting antisera were used to identify the same 
class of antibody at low levels in normal human serum. The 
new class, called IgD, has a serum concentration of 30 |JLg/ml 
and constitutes about 0.2% of the total immunoglobulin in 
serum. IgD, together with IgM, is the major membrane- 
bound immunoglobulin expressed by mature B cells, and its 
role in the physiology of B cells is under investigation. No bi- 
ological effector function has been identified for IgD. 



Antigenic Determinants 
on Immunoglobulins 



Since antibodies are glycoproteins, they 
is potent immunogens to 



themselves func- 
itibody response. 
Such anti-Ig antibodies are powerful tools for the study of 
B-cell development and humoral immune responses. The 
antigenic determinants, or epitopes, on immunoglobulin 
molecules fall into three major categories: isotypic, allotypic, 
and idiotypic determinants, which are located in characteris- 
tic portions of the molecule (Figure 4-17). 

Isotype 

Isotypic determinants are constant-region determinants that 
collectively define each heavy-chain class and subclass and 



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Antibodies: Structure and Functioi 



(a) Isotypic determinants 




Mouse IgGl Mouse IgM 

(b) Allotypic determinants 




(c) Idiotypic determinants 




For 



_| Antigi 

each type of determinant, the general location of determinants within 
the antibody molecule is shown (left) and two examples are illus- 
trated (center and right), (a) Isotypic determinants are constant- 
region determinants that distinguish each Ig class and subclass 
within a species, (b) Allotypic determinants are subtle amino acid 
differences encoded by different alleles of isotype genes. Allotypic 
differences can be detected by comparing the same antibody class 
among different inbred strains, (c) Idiotypic determinants are gen- 
erated by the conformation of the amino acid sequences of the 
heavy- and light-chain variable regions specific for each antigen. Each 
individual determinant is called an idiotope, and the sum of the indi- 
vidual idiotopes is the idiotype. 



each light-chain type and subtype within a species (see Fig- 
ure 4- 17a). Each isotype is encoded by a separate constant- 
region gene, and all members of a species carry the same 
constant-region genes (which may include multiple alleles). 
Within a species, each normal individual will express all iso- 
types in the serum. Different species inherit different con- 
stant-region genes and therefore express different isotypes. 
Therefore, when an antibody from one species is injected 
into another species, the isotypic determinants will be recog- 
nized as foreign, inducing an antibody response to the iso- 
typic determinants on the foreign antibody. Anti-isotype 



antibody is routinely used for research purposes to deter- 
mine the class or subclass of serum antibody produced dur- 
ing an immune response or to characterize the class of 
membrane-bound antibody present on B cells. 

Allotype 

Although all members of a species inherit the same set of iso- 
type genes, multiple alleles exist for some of the genes (see 
Figure 4-17b). These alleles encode subtle amino acid differ- 
ences, called allotypic determinants, that occur in some, but 
not all, members of a species. The sum of the individual allo- 
typic determinants displayed by an antibody determines its 
allotype. In humans, allotypes have been characterized for 
all four IgG subclasses, for one IgA subclass, and for the k 
light chain. The "y-chain allotypes are referred to as Gm 
markers. At least 25 different Gm allotypes have been identi- 
fied; they are designated by the class and subclass followed by 
the allele number, for example, Glm(l), G2m(23), G3m(ll), 
G4m(4a). Of the two IgA subclasses, only the IgA2 sub- 
class has allotypes, as A2m(l) and A2m(2). The k light 
chain has three allotypes, designated Km(l), Km(2), and 
Km(3). Each of these allotypic determinants represents dif- 
ferences in one to four amino acids that are encoded by 
different alleles. 

Antibody to allotypic determinants can be produced by 
injecting antibodies from one member of a species into an- 
other member of the same species who carries different allo- 
typic determinants. Antibody to allotypic determinants 
sometimes is produced by a mother during pregnancy in re- 
sponse to paternal allotypic determinants on the fetal im- 
munoglobulins. Antibodies to allotypic determinants can 
also arise from a blood transfusion. 

Idiotype 

The unique amino acid sequence of the V H and V L domains 
of a given antibody can function not only as an antigen-bind- 
ing site but also as a set of antigenic determinants. The idio- 
typic determinants arise from the sequence of the heavy- and 
light-chain variable regions. Each individual antigenic deter- 
minant of the variable region is referred to as an idiotope 
(see Figure 4- 17c). In some cases an idiotope may be the ac- 
tual antigen-binding site, and in some cases an idiotope may 
comprise variable-region sequences outside of the antigen- 
binding site. Each antibody will present multiple idiotopes; 
the sum of the individual idiotopes is called the idiotype of 
the antibody. 

Because the antibodies produced by individual B cells de- 
rived from the same clone have identical variable-region se- 
quences, they all have the same idiotype. Anti-idiotype 
antibody is produced by injecting antibodies that have mini- 
mal variation in their isotypes and allotypes, so that the idio- 
typic difference can be recognized. Often a homogeneous 
antibody such as myeloma protein or monoclonal antibody 
is used. Injection of such an antibody into a recipient who is 



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Generation of B-Cell and T-Cell Respor 



etically identical to the donor will result in the formation 
nti-idiotype antibody to the idiotypic determinants. 



The B-Cell Receptor 



Immunologists have long been puzzled about how mlg me- 
diates an activating signal after contact with an antigen. The 
dilemma is that all isotypes of mlg have very short cytoplas- 
mic tails: the mlgM and mlgD cytoplasmic tails contain only 
3 amino acids; the mlgA tail, 14 amino acids; and the mlgG 
and mlgE tails, 28 amino acids. In each case, the cytoplasmic 
tail is too short to be able to associate with intracellular sig- 
naling molecules (e.g., tyrosine kinases and G proteins). 

The answer to this puzzle is that mlg does not constitute 
the entire antigen-binding receptor on B cells. Rather, the B- 
cell receptor (BCR) is a transmembrane protein complex 
composed of mlg and disulfide-linked heterodimers called 
Ig-a/Ig-p. Molecules of this heterodimer associate with an 
mlg molecule to form a BCR (Figure 4-18). The Ig-a chain 










' General structure of the B-cell receptor (BCR). This 
antigen-binding receptor is composed of membrane-bound im- 
munoglobulin (mlg) and disulfide-linked heterodimers called 
lg-a/lg-p. Each heterodimer contains the immunoglobulin-fold 
structure and cytoplasmic tails much longer than those of mlg. 
As depicted, an mlg molecule is associated with one lg-ct/lg-(3 
heterodimer. [Adapted from A. D. Keegan and W. E. Paul, 1992, Im- 
munol. Today 13:63, and M. Reth, 1992, Annu. Rev. Immunol. 70:97.] 



has a long cytoplasmic tail containing 61 amino acids; the tail 
of the Ig-(3 chain contains 48 amino acids. The tails in both 
Ig-a and Ig-(3 are long enough to interact with intracellular 
signaling molecules. Discovery of the Ig-ot/Ig-(3 heterodimer 
by Michael Reth and his colleagues in the early 1990s has 
substantially furthered understanding of B-cell activation, 
which is discussed in detail in Chapter 11. 

Fc Receptors Bond to Fc Regions 
of Antibodies 

Many cells feature membrane glycoproteins called Fc recep- 
tors (FcR) that have an affinity for the Fc portion of the anti- 
body molecule. These receptors are essential for many of the 
biological functions of antibodies. Fc receptors are responsi- 
ble for the movement of antibodies across cell membranes 
and the transfer of IgG from mother to fetus across the pla- 
centa. These receptors also allow passive acquisition of anti- 
body by many cell types, including B and T lymphocytes, 
neutrophils, mast cells, eosinophils, macrophages, and nat- 
ural killer cells. Consequently, Fc receptors provide a means 
by which antibodies — the products of the adaptive immune 
system — can recruit such key cellular elements of innate im- 
munity as macrophages and natural killer cells. Engagement 
of antibody-bound antigens by the Fc receptors of macro- 
phages or neutrophils provides an effective signal for the 
efficient phagocytosis (opsonization) of antigen-antibody 
complexes. In addition to triggering such effector functions 
as opsonization or ADCC, crosslinking of Fc receptors by 
antigen-mediated crosslinking of FcR-bound antibodies can 
generate immunoregulatory signals that affect cell activation, 
induce differentiation and, in some cases, downregulate cel- 
lular responses. 

There are many different Fc receptors (Figure 4-19). The 
poly Ig receptor is essential for the transport of polymeric 
immunoglobulins (polymeric IgA and to some extent, pen- 
tameric IgM) across epithelial surfaces. In humans, the 
neonatal Fc receptor (FcR N ) transfers IgGs from mother to 
fetus during gestation and also plays a role in the regulation 
of IgG serum levels. Fc receptors have been discovered for all 
of the Ig classes. Thus there is an FcaR receptor that binds 
IgA, an FceR that binds IgE (see Figure 4-16 also), an FcSR 
that binds IgD, IgM is bound by an Fc|xR, and several vari- 
eties of FC7R receptors capable of binding IgG and its sub- 
classes are found in humans. In many cases, the crosslinking 
of these receptors by binding of antigen-antibody complexes 
results in the initiation of signal-transduction cascades that 
result in such behaviors as phagocytosis or ADCC. The Fc re- 
ceptor is often part of a signal-transducing complex that in- 
volves the participation of other accessory polypeptide 
chains. As shown in Figure 4-19, this may involve a pair of y 
chains or, in the case of the IgE receptor, a more complex as- 
semblage of two y chains and a p chain. The association of an 
extracellular receptor with an intracellular signal-transduc- 
ing unit was seen in the B cell receptor (Figure 4-18) and is a 
central feature of the T-cell-receptor complex (Chapter 9). 



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Antibodies: Structure and Functioi 




The Fc-binding polypeptides are shown in blue and, where present, dicated in the figure, many have been assigned CD designations (for 

accessory signal-transducing polypeptides are shown in green. The clusters of differentiation; see Appendix). [Adapted from M. Daeron, 

loops in these structures represent portions of the molecule with 1999, in The Antibodies, vol. 5, p. 53. Edited by M. Zanetti and]. D. 



The Immunoglobulin Superfamily 

The structures of the various immunoglobulin heavy and 
light chains described earlier share several features, suggest- 
ing that they have a common evolutionary ancestry. In 
particular, all heavy- and light-chain classes have the 
immunoglobulin-fold domain structure (see Figure 4-7). 
The presence of this characteristic structure in all im- 
munoglobulin heavy and light chains suggests that the genes 
encoding them arose from a common primordial gene en- 
coding a polypeptide of about 1 10 amino acids. Gene dupli- 
cation and later divergence could then have generated the 
various heavy- and light-chain genes. 

Large numbers of membrane proteins have been shown to 
possess one or more regions homologous to an im- 
munoglobulin domain. Each of these membrane proteins is 
classified as a member of the immunoglobulin superfamily. 
The term superfamily is used to denote proteins whose corre- 
sponding genes derived from a common primordial gene en- 
coding the basic domain structure. These genes have evolved 
independently and do not share genetic linkage or function. 
The following proteins, in addition to the immunoglobulins 
themselves, are representative members of the immunoglob- 
ulin superfamily (Figure 4-20): 



Ig-a/Ig-p heterodimer, part of the B-cell receptor 

Poly-Ig receptor, which contributes the secretory 
component to secretory IgA and IgM 

T-cell receptor 

T-cell accessory proteins, including CD2, CD4, CD8, 
CD28, and the y, 8, and e chains of CD 3 

Class I and class II MHC molecules 



microglobulin. 

s I MHC molecules 



protein associated with 



■ Various cell-adhesion molecules, including VCAM-1, 
ICAM-1, ICAM-2, and LFA-3 

■ Platelet-derived growth factor 

Numerous other proteins, some of them discussed in other 
chapters, also belong to the immunoglobulin superfamily. 

X-ray crystallographic analysis has not been accom- 
plished for all members of the immunoglobulin superfamily. 
Nevertheless, the primary amino acid sequence of these 
proteins suggests that they all contain the typical immuno- 
globulin-fold domain. Specifically, all members of the 
immunoglobulin superfamily contain at least one or more 
stretches of about 110 amino acids, capable of arrangement 



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8536d_ch04_076-104 9/5/02 



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Generation of B-Cell and T-Cell Respor 




nrn y y y y y y y y 

U A A A A A A A A A A A 



Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y 
A A A A A A A A A A A A A A A A A A A A ■ 



Y Y Y Y Y 
A A A A A 



Adhesion molecules 



Si* 



YHH — Y" 



T-cell accessory proteins 







YYl Y Y Y Y Y — Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y u ^ Y Y Y Y Y Y Y Y — Y Y Y Y Y Y 

LAI AAA A A A A A A A A A A A A A A A A A n A A A A A A A A A A A A A A 





Y Y Y Y 
A A A A 



rrn i ? 



Some members of the immunoglobulin superfamily, teins. I 

rturally related, usually membrane-bound glycopro- boxyl-tf 



c^ > 5 



:ases shown here except for p 2 -microglobulir 

al end of the molecule is anchored in the men 



into pleated sheets of antiparallel (3 strands, usually with an found in so many membrane proteins must have some func- 

invariant intrachain disulfide bond that closes a loop span- tion other than antigen binding. One possibility is that the 

ning 50-70 residues. immunoglobulin fold may facilitate interactions between 

Most members of the immunoglobulin superfamily can- membrane proteins. As described earlier, interactions can 

not bind antigen. Thus, the characteristic Ig-fold structure occur between the faces of |3 pleated sheets both of homolo- 



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8536d_ch04_076-104 9/5/02 6:19 AM Page 99 mac76 n 



loglobulin domains (e.g., C H 2/C H 2 interaction) 
and of nonhomologous domains (e.g., V H /V L and C H 1/C L 
interactions). 



Monoclonal Antibodies 

As noted in Chapter 3, most antigens offer multiple epitopes 
and therefore induce proliferation and differentiation of a 
variety of B-cell clones, each derived from a B cell that recog- 
nizes a particular epitope. The resulting serum antibodies are 
heterogeneous, comprising a mixture of antibodies, each 
specific for one epitope (Figure 4-21). Such a polyclonal an- 
tibody response facilitates the localization, phagocytosis, and 
complement-mediated lysis of antigen; it thus has clear ad- 



vantages for the organism in vivo. Unfortunately, the anti- 
body heterogeneity that increases immune protection in vivo 
often reduces the efficacy of an antiserum for various in vitro 
uses. For most research, diagnostic, and therapeutic pur- 
poses, monoclonal antibodies, derived from a single clone 
and thus specific for a single epitope, are preferable. 

Direct biochemical purification of a monoclonal anti- 
body from a polyclonal antibody preparation is not feasible. 
In 1975, Georges Kohler and Cesar Milstein devised a 
method for preparing monoclonal antibody, which quickly 
became one of immunology's key technologies. By fusing a 
normal activated, antibody-producing B cell with a myeloma 
cell (a cancerous plasma cell), they were able to generate a hy- 
brid cell, called a hybridoma, that possessed the immortal- 
growth properties of the myeloma cell and secreted the 



|BgP^l VISUALIZING CONCEPTS 



lOi Oi |Q 







rial antibodies 



" The conventional polyclonal antiserum produced which is derived from a single plasma cell, is specific for one epi- 

in response to a complex antigen contains a mixture of mono- tope on a complex antigen. The outline of the basic method for 

clonal antibodies, each specific for one of the four epitopes obtaining a monoclonal antibody is illustrated here, 
shown on the antigen (inset). In contrast, a monoclonal antibody, 



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Generation of B-Cell and T-Cell Respor 



antibody produced by the B cell (see Figure 4-2 1 ) . The result- 
ing clones of hybridoma cells, which secrete large quantities 
of monoclonal antibody, can be cultured indefinitely. The 
development of techniques for producing monoclonal anti- 
bodies, the details of which are discussed in Chapter 23, gave 
immunologists a powerful and versatile research tool. The 
significance of the work by Kohler and Milstein was ac- 
knowledged when each was awarded a Nobel Prize. 

Monoclonal Antibodies Have Important 
Clinical Uses 

Monoclonal antibodies are proving to be very useful as diag- 
nostic, imaging, and therapeutic reagents in clinical medi- 



±2Sf 




Immunotoxin 



Immunotoxin 



,Tumor-specific 







' (a) Toxins used to prepare immunotoxins include 
ricin, Shigella toxin, and diphtheria toxin. Each toxin contains an in- 
hibitory toxin chain (red) and a binding component (yellow). To make 
an immunotoxin, the binding component of the toxin is replaced 
with a monoclonal antibody (blue), (b) Diphtheria toxin binds to a 
cell-membrane receptor (left) and a diphtheria-immunotoxin binds 
to a tumor-associated antigen (right). In either case, the toxin is in- 
ternalized in an endosome. The toxin chain is then released into the 
cytoplasm, where it inhibits protein synthesis by catalyzing the inac- 
tivation of elongation factor 2 (EF-2). 



cine. Initially, monoclonal antibodies were used primarily as 
in vitro diagnostic reagents. Among the many monoclonal 
antibody diagnostic reagents now available are products for 
detecting pregnancy, diagnosing numerous pathogenic mi- 
croorganisms, measuring the blood levels of various drugs, 
matching histocompatibility antigens, and detecting anti- 
gens shed by certain tumors. 

Radiolabeled monoclonal antibodies can also be used in 
vivo for detecting or locating tumor antigens, permitting ear- 
lier diagnosis of some primary or metastatic tumors in pa- 
tients. For example, monoclonal antibody to breast-cancer 
cells is labeled with iodine- 131 and introduced into the blood 
to detect the spread of a tumor to regional lymph nodes. This 
monoclonal imaging technique can reveal breast-cancer 
t would be undetected by other, less sensitive 

Immunotoxins composed of tumor-specific monoclonal 
antibodies coupled to lethal toxins are potentially valuable 
therapeutic reagents. The toxins used in preparing immuno- 
toxins include ricin, Shigella toxin, and diphtheria toxin, all 
of which inhibit protein synthesis. These toxins are so potent 
that a single molecule has been shown to kill a cell. Each of 
these toxins consists of two types of functionally distinct 
polypeptide components, an inhibitory (toxin) chain and 
one or more binding chains, which interact with receptors on 
cell surfaces; without the binding polypeptide(s) the toxin 
cannot get into cells and therefore is harmless. An immuno- 
toxin is prepared by replacing the binding polypeptide(s) 
with a monoclonal antibody that is specific for a particular 
tumor cell (Figure 4-22a). In theory, the attached mono- 
clonal antibody will deliver the toxin chain specifically to tu- 
mor cells, where it will cause death by inhibiting protein 
synthesis (Figure 4-22b). The initial clinical responses to 
such immunotoxins in patients with leukemia, lymphoma, 
and some other types of cancer have shown promise, and re- 
search to develop and demonstrate their safety and effective- 
ness is underway. 

Abzymes Are Monoclonal Antibodies 
That Catalyze Reactions 

The binding of an antibody to its antigen is similar in many 
ways to the binding of an enzyme to its substrate. In both 
cases the binding involves weak, noncovalent interactions 
and exhibits high specificity and often high affinity. What 
distinguishes an antibody-antigen interaction from an en- 
zyme-substrate interaction is that the antibody does not alter 
the antigen, whereas the enzyme catalyzes a chemical change 
in its substrate. However, like enzymes, antibodies of appro- 
priate specificity can stabilize the transition state of a bound 
substrate, thus reducing the activation energy for chemical 
modification of the substrate. 

The similarities between antigen-antibody interactions 
and enzyme-substrate interactions raised the question of 
whether some antibodies could behave like enzymes and 
catalyze chemical reactions. To investigate this possibility, a 



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Antibodies: Structure and Functioi 



hapten-carrier complex was synthesized in which the hapten 
structurally resembled the transition state of an ester under- 
going hydrolysis. Spleen cells from mice immunized with this 
transition state analogue were fused with myeloma cells to 
generate monoclonal antihapten monoclonal antibodies. 
When these monoclonal antibodies were incubated with an 
ester substrate, some of them accelerated hydrolysis by about 
1000-fold; that is, they acted like the enzyme that normally 
catalyzes the substrate's hydrolysis. The catalytic activity of 
these antibodies was highly specific; that is, they hydrolyzed 
only esters whose transition-state structure closely resembled 
the transition state analogue used as a hapten in the immu- 
nizing conjugate. These catalytic antibodies have been called 
abzymes in reference to their dual role as antibody and 
enzyme. 

A central goal of catalytic antibody research is the deriva- 
tion of a battery of abzymes that cut peptide bonds at specific 
amino acid residues, much as restriction enzymes cut DNA 
at specific sites. Such abzymes would be invaluable tools in 
the structural and functional analysis of proteins. Addition- 
ally, it may be possible to generate abzymes with the ability to 
dissolve blood clots or to cleave viral glycoproteins at specific 
sites, thus blocking viral infectivity. Unfortunately, catalytic 
antibodies that cleave the peptide bonds of proteins have 
been exceedingly difficult to derive. Much of the research 
currently being pursued in this field is devoted to the solu- 
tion of this important but difficult problem. 



SUMMARY 

■ An antibody molecule consists of two identical light chains 
and two identical heavy chains, which are linked by disul- 
fide bonds. Each heavy chain has an amino -terminal vari- 
able region followed by a constant region. 

■ In any given antibody molecule, the constant region con- 
tains one of five basic heavy-chain sequences ( (jl, -y, 8, a, or 
e) called isotypes and one of two basic light-chain se- 
quences (k or X) called types. 

■ The heavy-chain isotype determines the class of an anti- 
body (|X, IgM; 7, IgG; 8, IgD; a, IgA; and e, IgE). 

■ The five antibody classes have different effector functions, 
5, and half-lives. 



i Each of the domains in the immunoglobulin molecule has 
a characteristic tertiary structure called the immunoglob- 
ulin fold. The presence of an immunoglobulin fold do- 
main also identifies many other nonantibody proteins as 
members of the immunoglobulin superfamily. 

i Within the amino-terminal variable domain of each heavy 
and light chain are three complementarity-determining re- 
gions (CDRs). These polypeptide regions contribute the anti- 
gen-binding site of an antibody, determining its specificity. 

i Immunoglobulins are expressed in two forms: secreted 
antibody that is produced by plasma cells, and mem- 
brane-bound antibody that associates with Ig-a/Ig-fj 



heterodimers to form the B-cell antigen receptor present 
on the surface of B cells. 

■ The three major effector functions that enable antibodies 
to remove antigens and kill pathogens are: opsonization, 
which promotes antigen phagocytosis by macrophages 
and neutrophils; complement activation, which activates a 
pathway that leads to the generation of a collection of pro- 
teins that can perforate cell membranes; and antibody- 
dependent cell-mediated cytotoxicity (ADCC), which can 
kill antibody-bound target cells. 

■ Unlike polyclonal antibodies that arise from many B cell 
clones and have a heterogeneous collection of binding 
sites, a monoclonal antibody is derived from a single B cell 
clone and is a homogeneous collection of binding sites. 



References 

Frazer, J. K., and J. D. Capra. 1999. Immunoglobulins: structure 
and function. In Funda ''logy, 4th ed. W. E. Paul, 

ed. Philadelphia, Lippincott-Raven. 

Kohler, G., and C. Milstein. 1975. Continuous cultures of fused 
cells secreting antibody of predefined specificity. Nature 
256:495. 

Kraehenbuhl, J. P., and M. R. Neutra. 1992. Transepithelial trans- 
port and mucosal defence II: secretion of IgA. Trends Cell Biol. 
2:134. 

Immunology Today, The Immune Receptor Supplement, 2nd ed. 
1997. Elsevier Trends Journals, Cambridge, UK (ISSN 1365- 
1218). 



Newman, J. 1995. How bre 
273(6):76. 



milk protects newbon 

pie. 



Reth, M. 1995. The B-cell antigen receptc 
ceptor. Immunol. Today 16:310. 

Stanfield, R. L., and I. A. Wilson. 1995. Protein-peptide interac- 
tions. Curr. Opin. Struc. Biol. 5:103. 

Wedemayer, G. J., P. A. Patten, L. H. Wang, P. G Schultz, and 
R. C. Stevens. 1997. Structural insights into the evolution of 
an antibody combining site. Science, 276:1665. 

Wentworth, P., and Janda, K. 1998. Catalytic Antibodies. Curr. 
Opin. Chem. Biol. 8:138. 

Wilson, I. A., and R. L. Stanfield. 1994. Antibody-antibody inter- 
actions: new structures and new conformational changes. 
Curr. Opin. Struc. Biol. 4:857. 



USEFULWEB SITES 



http://in 



nuno.bme.n 



j.edu/ 



The Kabat Database of Sequences of Proteins of Immunolog- 
ical Interest: This site has the amino acid and DNA sequences 
of many antibodies and other proteins that play important 
roles in immunology. 



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Generation of B-Cell and T-Cell Respor 



http://w 



c.uk/~martin/abs 



Antibodies — Structure and Sequence: This Web site summa- 
rizes useful information on antibody structure and sequence. 
It provides general information on antibodies and crystal 
s and links to other antibody-related information. 

ilm.nih.gov 

National Center for Biotechnology Information (NCBI): A 
unique and comprehensive resource of computerized data- 
bases of bibliographic information, nucleic acid sequences, 
protein sequences, and sequence analysis tools created and 
maintained by the National Library of Medicine. 



http://w 



http://w 



w.ncbi.r 



n. nih.gov/Structure/ 



The Molecular Modeling Database (MMDB) contains 3-di- 
mensional structures determined by x-ray crystallography 
and NMR spectroscopy. The data for MMDB are obtained 
from the Protein Data Bank (PDB). The National Center for 
Biotechnology Information (NCBI) has structural data 
crosslinked to bibliographic information, to databases of pro- 
tein and nucleic acid sequences, and to the NCBI animal tax- 
onomy database. The NCBI has developed a 3D structure 
viewer, Cn3D, for easy interactive visualization of molecular 



http://w 



iass.edu/microbio/chime/explorer/ 

Protein Explorer is a molecular visualization program created 
by Eric Martz with the support of the National Science Foun- 
dation to make it easier for students, educators, and scientists 
to use interactive and dynamic molecular visualization tech- 
niques. Many will find it easier to use than Chime and Rasmol. 



http://imgt.ci 



s.fr 



IMGT, the international ImMunoGeneTics database created 
by Marie-Paule Lefranc, is a well organized, powerful, and 
comprehensive information system that specializes in im- 
munoglobulins, T-cell receptors and major histocompatibil- 
ity complex (MHC) molecules of all vertebrate species. 



Study Questions 



Clinical Focus Question Two pharmaceutical companies 
make IVIG. Company A produces their product from pools of 
100,000 donors drawn exclusively from the population of the 
United States. Company B makes their IVIG from pools of 
60,000 donors drawn in equal numbers from North America, 
Europe, Brazil, and Japan. 

a. Which product would you expect to have the broadest 
spectrum of pathogen reactivities? Why? 

b. Assume the patients receiving the antibody will (1) never 
leave the USA, or (2) travel extensively in many parts of the 
world. Which company's product would you choose for 
each of these patient groups? Justify your choices. 

1 . Indicate whether each of the following statements is true or 
false. If you think a statement is false, explain why. 

a. A rabbit immunized with human IgG3 will produce anti- 
body that reacts with all subclasses of IgG in humans. 



Go to www.whfreeman.ee 
Review and quiz of key te 



<$s 



b. All immunoglobulin molecules on the surface of a given 
B cell have the same idiotype. 

c. All immunoglobulin molecules on the surface of a given 
B cell have the same isotype. 

d. All myeloma protein molecules derived from a single 
myeloma clone have the same idiotype and allotype. 

e. Although IgA is the major antibody species that under- 
goes transcytosis, polymeric IgM, but : 
IgA, can also undergo transcytosis. 

f. The hypervariable regions make signific; 
the epitope. 

g. IgG functions more effectively than IgM in bacterial ag- 






h. Although monoclonal antibodies are often preferred for 
research and diagnostic purposes, both monoclonal and 
polyclonal antibodies can be highly specific. 

i. All isotypes are normally found in each individual of a 

j. The heavy-chain variable region (V H ) is twice as long as 
the light-chain variable region (V L ). 

2. You are an energetic immunology student who has isolated 
protein X, which you believe is a new isotype of human im- 
munoglobulin. 

a. What structural features would protein X have to have in 
order to be classified as an immunoglobulin? 

b. You prepare rabbit antisera to whole human IgG, human 
k chain, and human 7 chain. Assuming protein X is, in 
fact, a new immunoglobulin isotype, to which of these 
antisera would it bind? Why? 

c. Devise an experimental procedure for preparing an anti- 
serum that is specific for protein X. 

3. According to the clonal selection theory, all the im- 
munoglobulin molecules on a single B cell have the same 
antigenic specificity. Explain why the presence of both IgM 
and IgD on the same B cell does not violate the unispecificity 
implied by clonal selection. 



4. IgG, which contains 7 heavy chains, developed much more 
recently during evolution than IgM, which contains u, heavy 
chains. Describe two advantages and two disadvantages that 
IgG has in comparison with IgM. 

5. Although the five immunoglobulin isotypes share many 
common structural features, the differences in their struc- 
tures affect their biological activities. 

a. Draw a schematic diagram of a typical IgG molecule and 
label each of the following parts: H chains, L chains, in- 
terchain disulfide bonds, intrachain disulfide bonds, 
hinge, Fab, Fc, and all the domains. Indicate which do- 
mains are involved in antigen binding. 

b. How would you have to modify the diagram of IgG to de- 
pict an IgA molecule isolated from saliva? 

c. How would you have to modify the diagram of IgG to de- 
pict serum IgM? 

6. Fill out the accompanying table relating to the properties of 
IgG molecules and their various parts. Insert a (+) if the 
molecule or part exhibits the property; a ( — ) if it does not; 
and a ( + / — ) if it does so only weakly. 



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al . / Immunology 5e : 



Whole H L 
Property IgG chain chain Fab F(ab') 2 Fc 


Binds antigen 














Bivalent 














Binds to Fc 
receptors 














in presence 
of antigen 














HasV 
domains 














HasC 
domains 















9. The characteristic 

termed the immunoglobulin fold, also o< 
ous membrane proteins belonging to th 
superfamily. 

a. Describe the typical features that define the im- 
munoglobulin-fold domain structure. 

b. Consider proteins that belong to the immunoglobulin 
superfamily. What do all of these proteins have in com- 
mon? Describe two different Ig superfamily members 
that bind antigen. Identify four different Ig superfamily 
members that do not bind antigen. 



. Where are the CDR regions located o: 
and what are their functions? 



n antibody molecule 



ino acid sequence at each position in a 
n be expressed by a quantity termed the 
the largest and smallest values of vari- 



1 1 . The a 

polypeptide chain c 
variability. What ar 
ability possible? 

12. You prepare an immunotoxin by conjugating diphther 
toxin with a monoclonal antibody specific for a tum( 



7. Because immunoglobulin molecules possess antigenic de- 
terminants, they themselves can function as immunogens, 
inducing formation of antibody. For each of the following 
immunization scenarios, indicate whether anti-immuno- 
globulin antibodies would be formed to isotypic (IS), allo- 
typic (AL), or idiotypic (ID) determinants: 

a. Anti-DNP antibodies produced in a BALB/c mouse are 
injected into a C57BL/6 mouse. 

b. Anti-BGG monoclonal antibodies from a BALB/c mouse 
are injected into another BALB/c mouse. 

c. Anti-BGG antibodies produced in a BALB/c mouse are 
injected into a rabbit. 

d. Anti-DNP antibodies produced in a BALB/c mouse are 
injected into an outbred mouse. 

e. Anti-BGG antibodies produced in a BALB/c mouse are 
injected into the same mouse. 

8. Write YES or NO in the accompanying table to indicate 
whether the rabbit antisera listed at the top react with the 
mouse antibody components listed at the left. 



y K IgG Fab IgG Fc J 
chain chain fragment fragment chain 


Mouse 












Mouse 












Mouse 
IgM whole 












Mouse 
IgM Fc 
fragment 













a. If this immunotoxin is injected into an animal, will any 
normal cells be killed? Explain. 

b. If the antibody part of the immunotoxin is degraded so 
that the toxin is released, will normal cells be killed? Ex- 
plain. 

1 3. An investigator wanted to make a rabbit antiserum specific 
for mouse IgG. She injected a rabbit with purified mouse 
IgG and obtained an antiserum that reacted strongly with 
mouse IgG. To her dismay, however, the antiserum also re- 
acted with each of the other mouse isotypes. Explain why she 
got this result. How could she make the rabbit antiserum 
specific for mouse IgG? 

14. You fuse spleen cells having a normal genotype for im- 
munoglobulin heavy chains (H) and light chains (L) with 
three myeloma-cell preparations differing in their im- 
munoglobulin genotype as follows: (a) H + , L + ; (b) H~, L + ; 
and (c) H~, L~. For each hybridoma, predict how many 
unique antigen-binding sites, composed of one H and one L 
chain, theoretically could be produced and show the chain 
structure of the possible antibody molecules. For each possi- 
ble antibody molecule indicate whether the chains would 
originate from the spleen (S) or from the myeloma (M) fu- 
sion partner (e.g., H s L s /H m L m ). 

15. For each immunoglobulin isotype (a-e) select the descrip- 
tion^) listed below (1-12) that describe that isotype. Each 
description may be used once, more than once, or not at all; 
more than one description may apply to some isotypes. 






-IgE 
_IgG 



Descriptions 

( 1 ) Secreted form is a pen tamer of the bas 

(2) Binds to Fc receptors on mast cells 



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(3) Multimeric forms have a J chain 

(4) Present on the surface of mature, unprimed B cells 

(5) The most abundant isotype in serum 

(6) Major antibody in secretions such as saliva, tears, and 
breast milk 

(7) Present on the surface of immature B cells 

(8) The first serum antibody made in a primary immune 
response 

(9) Plays an important role in immediate hypersensitivity 

(10) Plays primary role in protecting against pathogens that 
invade through the gut or respiratory mucosa 

(11) Multimeric forms may contain a secretory component 

(12) Least abundant isotype in serum 



1 6. Describe four distinct roles played by Fc receptors. In what 
ways is signal transduction from Fc receptors similar to sig- 
nal transduction from the B-cell receptor? 

1 7. What is IVIG and what are some of the mechanisms by 
which it might protect the body against infection? Suppose 
one had the option of collecting blood for the manufacture 
of IVIG from the following groups of healthy individuals: 
35-year-old men who had lived all of their lives in isolated 
villages in the mountains of Switzerland, or 45-55-year-old 
men who had been international airline pilots for 20 years. 
Which group would provide the better pool of blood? Justify 
your answer. 



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Organization and 
Expression of 
Immunoglobulin 
Genes 



CNE OF THE MOST REMARKABLE FEATURES OF 
the vertebrate immune system is its ability to 
respond to an apparently limitless array of for- 
eign antigens. As immunoglobulin (Ig) sequence data accu- 
mulated, virtually every antibody molecule studied was 
found to contain a unique amino acid sequence in its vari- 
able region but only one of a limited number of invariant se- 
quences in its constant region. The genetic basis for this 
combination of constancy and tremendous variation in a 
single protein molecule lies in the organization of the im- 
munoglobulin genes. 

In germ-line DNA, multiple gene segments encode por- 
tions of a single immunoglobulin heavy or light chain. These 
gene segments are carried in the germ cells but cannot be 
transcribed and translated into complete chains until they 
are rearranged into functional genes. During B-cell matura- 
tion in the bone marrow, certain of these gene segments are 
randomly shuffled by a dynamic genetic system capable of 
generating more than 10 6 combinations. Subsequent 
processes increase the diversity of the repertoire of antibody 
binding sites to a very large number that exceeds 10 6 by at 
least two or three orders of magnitude. The processes of B- 
cell development are carefully regulated: the maturation of a 
progenitor B cell progresses through an ordered sequence of 
Ig-gene rearrangements, coupled with modifications to the 
gene that contribute to the diversity of the final product. By 
the end of this process, a mature, immunocompetent B cell 
will contain coding sequences for one functional heavy- 
chain variable-region and one light-chain variable-region. 
The individual B cell is thus antigenically committed to a 
specific epitope. After antigenic stimulation of a mature B 
cell in peripheral lymphoid organs, further rearrangement 
of constant-region gene segments can generate changes in 
the isotype expressed, which produce changes in the biolog- 
ical effector functions of the immunoglobulin molecule 
without changing its specificity. Thus, mature B cells contain 
chromosomal DNA that is no longer identical to germ-line 



chapter 5 



L V K J K J K C K 



Polyadenylatioi 
RNA splicing 



^ (A)„ 



■ Genetic Model Compatible with Ig Structure 

■ Multigene Organization of Ig Genes 

■ Variable-Region Gene Rearrangements 

■ Mechanism of Variable-Region DNA 
Rearrangements 

■ Generation of Antibody Diversity 

■ Class Switching among Constant-Region Genes 

■ Expression of Ig Genes 

■ Synthesis, Assembly, and Secretion of 
Immunoglobulins 

■ Regulation of Ig-Gene Transcription 

■ Antibody Genes and Antibody Engineering 



DNA. While we think of genomic DNA as a stable genetic 
blueprint, the lymphocyte cell lineage does not retain an in- 
tact copy of this blueprint. Genomic rearrangement is an es- 
sential feature of lymphocyte differentiation, and no other 
vertebrate cell type has been shown to undergo this process. 
This chapter first describes the detailed organization of 
the immunoglobulin genes, the process of Ig-gene rearrange- 
ment, and the mechanisms by which the dynamic im- 
munoglobulin genetic system generates more than 10 8 
different antigenic specificities. Then it describes the mecha- 
nism of class switching, the role of differential RNA process- 
ing in the expression of immunoglobulin genes, and the 
regulation of Ig-gene transcription. The chapter concludes 
with the application of our knowledge of the molecular 



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VISUALIZING CONCEPTS 

CELL 



organs 






Hematopoietic 

Lymphoid cell (CJ) None 

Partial heavy-chain gene rearrangement 
Pro-B cell (Q) None 

Complete heavy-chain gene rearrangement 
Pre-B cell \C_J/ [L Heavy chain + surrogate lis 

Immature B 

Change in RNA processing 

= Mature B cell " 

Activated B cell =jQjfli) 
Differentiation 

> IgM-secreting plasma cells 

i Class switching 

fi *) Plasma cells 
| iSOtyPCS 

Y Y Y 













Overview of B-cell development. The events that ripheral lymphoid organs require antigen. The labels mlgM and 
jr during maturation in the bone marrow do not require anti- mlgD refer to membrane-associated Igs. IgC, IgA, and IgE are se- 
whereas activation and differentiation of mature B cells in pe- creted immunoglobulins. 



biology of immunoglobulin genes to the engineering of anti- 
body molecules for therapeutic and research applications. 
Chapter 1 1 covers in detail the entire process of B-cell devel- 
opment from the first gene rearrangements in progenitor B 
cells to final differentiation into memory B cells and anti- 
body-secreting plasma cells. Figure 5-1 outlines the sequen- 
tial stages in B-cell development, many of which result from 
critical rearrangements. 



Genetic Model Compatible 
with Ig Structure 

The results of the immunoglobulin-sequencing studies de- 
scribed in Chapter 4 revealed a number of features of 
immunoglobulin structure that were difficult to reconcile 
with classic genetic models. Any viable model of the 



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' Immunology 5e: 



immunoglobulin genes had t 
properties of antibodies: 



for the following 



The vast diversity of antibody specificities 

The presence in Ig heavy and light chains of a variable 
region at the amino-terminal end and a constant region 
at the carboxyl-terminal end 

The existence of isotypes with the same antigenic 
specificity, which result from the association of a given 
variable region with different heavy- chain constant 
regions 



Germ-Line and Somatic-Variation Models 
Contended To Explain Antibody Diversity 

For several decades, immunologists sought to imagine a ge- 
netic mechanism that could explain the tremendous diversity 
of antibody structure. Two different sets of theories emerged. 
The germ-line theories maintained that the genome con- 
tributed by the germ cells, egg and sperm, contains a large 
repertoire of immunoglobulin genes; thus, these theories in- 
voked no special genetic mechanisms to account for anti- 
body diversity. They argued that the immense survival value 
of the immune system justified the dedication of a significant 
fraction of the genome to the coding of antibodies. In con- 
trast, the somatic-variation theories maintained that the 
genome contains a relatively small number of immunoglob- 
ulin genes, from which a large number of antibody specifici- 
ties are generated in the somatic cells by mutation or 
recombination. 

As the amino acid sequences of more and more im- 
munoglobulins were determined, it became clear that there 
must be mechanisms not only for generating antibody diver- 
sity but also for maintaining constancy. Whether diversity 
was generated by germ-line or by somatic mechanisms, a 
paradox remained: How could stability be maintained in the 
constant (C) region while some kind of diversifying mecha- 
nism generated the variable (V) region? 

Neither the germ-line nor the somatic-variation propo- 
nents could offer a reasonable explanation for this central 
feature of immunoglobulin structure. Germ-line proponents 
found it difficult to account for an evolutionary mechanism 
that could generate diversity in the variable part of the many 
heavy- and light-chain genes while preserving the constant 
region of each unchanged. Somatic-variation proponents 
found it difficult to conceive of a mechanism that could di- 
versify the variable region of a single heavy- or light- chain 
gene in the somatic cells without allowing alteration in the 
amino acid sequence encoded by the constant region. 

A third structural feature requiring an explanation 
emerged when amino acid sequencing of the human 
myeloma protein called Til revealed that identical variable- 
region sequences were associated with both y and |x heavy- 
chain constant regions. A similar phenomenon was observed 



in rabbits by C. Todd, who found that a particular allotypic 
marker in the heavy-chain variable region could be associ- 
ated with a, -y, and jjl heavy-chain constant regions. Consid- 
erable additional evidence has confirmed that a single 
variable-region sequence, defining a particular antigenic 
specificity, can be associated with multiple heavy-chain 
constant-region sequences; in other words, different classes, 
or isotypes, of antibody (e.g., IgG, IgM) can be expressed 
with identical variable-region sequences. 



Dreyer and Bennett Proposed 
the Two-Gene Model 

In an attempt to develop a genetic model consistent with the 
known findings about the structure of immunoglobulins, W. 
Dreyer and J. Bennett suggested, in their classic theoretical 
paper of 1965, that two separate genes encode a single im- 
munoglobulin heavy or light chain, one gene for the V region 
(variable region) and the other for the C region (constant re- 
gion). They suggested that these two genes must somehow 
come together at the DNA level to form a continuous mes- 
sage that can be transcribed and translated into a single Ig 
heavy or light chain. Moreover, they proposed that hundreds 
or thousands of V-region genes were carried in the germ line, 
whereas only single copies of C-region class and subclass 
genes need exist. 

The strength of this type of recombinational model 
(which combined elements of the germ-line and somatic- 
variation theories) was that it could account for those im- 
munoglobulins in which a single V region was combined 
with various C regions. By postulating a single constant- 
region gene for each immunoglobulin class and subclass, the 
model also could account for the conservation of necessary 
biological effector functions while allowing for evolutionary 
diversification of variable-region genes. 

At first, support for the Dreyer and Bennett hypothesis 
was indirect. Early studies of DNA hybridization kinetics us- 
ing a radioactive constant-region DNA probe indicated that 
the probe hybridized with only one or two genes, confirming 
the model's prediction that only one or two copies of each 
constant-region class and subclass gene existed. However, in- 
direct evidence was not enough to overcome stubborn resis- 
tance in the scientific community to the hypothesis of Dreyer 
and Bennet. The suggestion that two genes encoded a single 
polypeptide contradicted the existing one gene-one 
polypeptide principle and was without precedent in any 
known biological system. 

As so often is the case in science, theoretical and intellec- 
tual understanding of Ig-gene organization progressed ahead 
of the available methodology. Although the Dreyer and Ben- 
nett model provided a theoretical framework for reconciling 
the dilemma between Ig-sequence data and gene organiza- 
tion, actual validation of their hypothesis had to wait for sev- 
eral major technological advances in the field of molecular 
biology. 



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Generation of B-Cell and T-Cell Respor 



-Immunoglobulin 



Tonegawa's Bombshell- 
Genes Rearrange 

In 1976, S. Tonegawa and N. Hozumi found the first direct 
evidence that separate genes encode the V and C regions of 
immunoglobulins and that the genes are rearranged in the 
course of B-cell differentiation. This work changed the field 
of immunology. In 1987, Tonegawa was awarded the Nobel 
Prize for this work. 

Selecting DNA from embryonic cells and adult myeloma 
cells — cells at widely different stages of development — 
Tonegawa and Hozumi used various restriction endonucle- 
ases to generate DNA fragments. The fragments were then 
separated by size and analyzed for their ability to hybridize 
with a radiolabeled mRNA probe. Two separate restriction 
fragments from the embryonic DNA hybridized with the 
mRNA, whereas only a single restriction fragment of the 
adult myeloma DNA hybridized with the same probe. Tone- 
gawa and Hozumi suggested that, during differentiation of 
lymphocytes from the embryonic state to the fully differenti- 
ated plasma-cell stage (represented in their system by the 



myeloma cells), the V and C genes undergo rearrangement. 
In the embryo, the V and C genes are separated by a large 
DNA segment that contains a restriction-endonuclease site; 
during differentiation, the V and C genes are brought closer 
together and the intervening DNA sequence is eliminated. 

The pioneering experiments of Tonegawa and Hozumi 
employed a tedious and time-consuming procedure that has 
since been replaced by the much more powerful approach of 
Southern-blot analysis. This method, now universally used to 
investigate the rearrangement of immunoglobulin genes, 
eliminates the need to elute the separated DNA restriction 
fragments from gel slices prior to analysis by hybridization 
with an immunoglobulin gene segment probe. Figure 5-2 
shows the detection of rearrangement at the k light-chain lo- 
cus by comparing the fragments produced by digestion of 
DNA from a clone of B-lineage cells with the pattern ob- 
tained by digestion of non-B cells (e.g., sperm or liver cells). 
The rearrangement of a V gene deletes an extensive section of 
germ-line DNA, thereby creating differences between re- 
arranged and unrearranged Ig loci in the distribution and 
number of restriction sites. This results in the generation of 





/"Deleted 


RE RE RE RE RE 

V V 2 V, 

5 ' M II M II M /HZKZ>- 3 


Rearrangement 


Probe \ 




RE digestionX 





Germ line 


Rearranged 



an immunoglobulin locus. The number and size of restriction frag- 
ments generated by the treatment of DNA with a restriction enzyme 
is determined by the sequence of the DNA.The digestion of 
arranged DNA with a restriction enzyme (RE) yields a pattern of 
striction fragments that differ from those obtained by digestion of 
unrearranged locus with the same RE. Typically, the fragments are 
alyzed by the technique of Southern blotting. In this example, a probe 
that includes a J gene segment is used to identify RE digestion frag- 
ments that include all or portions of this segment. As show 
arrangement results in the deletion of a segment of germ-line DNA 
and the loss of the restriction sites that it includes. It also results in 
the joining of gene segments, in this case a V and a J segment, that 



are separated in the germ line. Consequently, fragments dependent 
on the presence of this segment for their generation are absent from 
the restriction-enzyme digest of DNA from the rearranged locus. Fur- 
thermore, rearranged DNA gives rise to novel fragments that are ab- 
sent from digests of DNA in the germ-line configuration. This can be 
useful because both B cells and non-B cells have two immunoglobu- 
lin loci. One ofthese is rearranged and the other is not. Consequently, 
unless a genetic accident has resulted in the loss of the germ-line lo- 
cus, digestion of DNA from a myeloma or normal B-cell clone will 
produce a pattern of restriction that includes all of those in a germ- 
line digest plus any novel fragments that are generated from the 
change in DNA sequence that accompanies rearrangement. Note 
that only one of the several J gene segements present is shown. 



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different restriction patterns by rearranged and unre- 
arranged loci. Extensive application of this approach has 
demonstrated that the Dreyer and Bennett two-gene 
model — one gene encoding the variable region and another 
encoding the constant region — applied to both heavy and 
light-chain genes. 



Multigene Organization of Ig Genes 

As cloning and sequencing of the light- and heavy-chain 
DNA was accomplished, even greater complexity was re- 
vealed than had been predicted by Dreyer and Bennett. The k 
and X light chains and the heavy chains are encoded by sepa- 
rate multigene families situated on different chromosomes 
(Table 5-1). In germ-line DNA, each of these multigene fam- 
ilies contains several coding sequences, called gene seg- 
ments, separated by noncoding regions. During B-cell 
maturation, these gene segments are rearranged and brought 
together to form functional immunoglobulin genes. 

Each Multigene Family Has Distinct Features 

The k and A. light-chain families contain V, J, and C gene seg- 
ments; the rearranged VJ segments encode the variable re- 
gion of the light chains. The heavy- chain family contains V, 
D, J, and C gene segments; the rearranged VDJ gene seg- 
ments encode the variable region of the heavy chain. In each 
gene family, C gene segments encode the constant regions. 
Each V gene segment is preceded at its 5' end by a small exon 
that encodes a short signal or leader (L) peptide that guides 
the heavy or light chain through the endoplasmic reticulum. 
The signal peptide is cleaved from the nascent light and heavy 
chains before assembly of the finished immunoglobulin mol- 
ecule. Thus, amino acids encoded by this leader sequence do 
not appear in the immunoglobulin molecule. 

X-CHAIN MULTIGENE FAMILY 

The first evidence that the light-chain variable region was ac- 
tually encoded by two gene segments appeared when Tone- 
gawa cloned the germ-line DNA that encodes the variable 
region of mouse X light chain and determined its complete 



segments, and four C x 
:udogene, a de- 
mg protein; such 



nucleotide sequence. When the nucleotide sequence was 
compared with the known amino acid sequence of the X- 
chain variable region, an unusual discrepancy was observed. 
Although the first 97 amino acids of the X-chain variable re- 
gion corresponded to the nucleotide codon sequence, the re- 
maining 13 carboxyl-terminal amino acids of the protein's 
variable region did not. It turned out that many base pairs 
away a separate, 39-bp gene segment, called J for joining, en- 
coded the remaining 13 amino acids of the X-chain variable 
region. Thus, a functional X variable-region gene contains 
two coding segments — a 5' V segment and a 3' J segment — 
which are separated by a noncoding DNA sequence in unre- 
arranged germ-line DNA. 

The X multigene family in t 
three V x gene segments, four J ; 
gene segments (Figure 5-3a). The J x 4 if 
fective gene that is incapable of e 
genes are indicated with the psi symbol (i|i). Interestingly, 
J x 4's constant region partner, C x 4, is a perfectly functional 
gene. The V x and the three functional J x gene segments en- 
code the variable region of the light chain, and each of the 
three functional C x gene segments encodes the constant re- 
gion of one of the three X-chain subtypes (XI, X2, and 
X3). In humans, the lambda locus is more complex. There 
are 31 functional V x gene segments, 4 J x segments, and 
7 C x segments. In additional to the functional gene seg- 
ments, the human lambda complex contains many V x , J x , 
and C x pseudogenes. 

k-CHAIN MULTIGENE FAMILY 

The K-chain multigene family in the mouse contains approx- 
imately 85 V K gene segments, each with an adjacent leader se- 
quence a short distance upstream (i.e., on the 5' side). There 
are five J K gene segments (one of which is a nonfunctional 
pseudogene) and a single C K gene segment (Figure 5-3b). As 
in the X multigene family, the V K and J K gene segments en- 
code the variable region of the k light chain, and the C K gene 
segment encodes the constant region. Since there is only one 
C K gene segment, there are no subtypes of k light chains. 
Comparison of parts a and b of Figure 5-3 shows that the 
arrangement of the gene segments is quite different in the k 
and X gene families. The K-chain multigene family in hu- 
mans, which has an organization similar to that of the 
mouse, contains approximately 40 V K gene segments, 5 J K 
segments, and a single C K segment. 



Might chain 
k Light chain 
Heavy chain 



CHROMOSOME 
Human Mouse 



HEAVY-CHAIN MULTIGENE FAMILY 

The organization of the immunoglobulin heavy-chain genes 
is similar to, but more complex than, that of the k and 
X light-chain genes (Figure 5-3c). An additional gene 
segment encodes part of the heavy-chain variable region. 
The existence of this gene segment was first proposed 
by Leroy Hood and his colleagues, who compared the 
heavy-chain variable-region amino acid sequence with the 
V H and J H nucleotide sequences. The V H gene segment was 
found to encode amino acids 1 to 94 and the J H gene segment 



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Generation of B-Cell and T-Cell Respor 



VISUALIZING CONCEPTS 



(a) A,-chain DNA 

LV X 2 h 2 C,2 ) X 4 C X 4 

^m — u-ed-q-ed-//- 

70 1.2 2.0 1.3 

kb kb kb kb 



hi C X 3 hi <*! 



(b) K-chain DNA 
L V..1 L V K 2 



4Hj^Hj^^3' 



:) Heavy-chain DNA 
B = ~134 
LV H 1 LV H n D H 1 D H 13 J H 1 

■V/-D- 



segments in the mouse: (a) X light chain, (b) k light chain, and (c) gene segments in 

heavy chain. The A. and k light chains are encoded by V, J, and C chain diagram, 
gene segments. The heavy chain is encoded by V, D, J, and C gene 



n kilobases (kb) separating the v 
germ-line DNA are shown belov 



was found to encode amino acids 98 to 1 13; however, neither 
of these gene segments carried the information to encode 
amino acids 95 to 97. When the nucleotide sequence was de- 
termined for a rearranged myeloma DNA and compared 
with the germ-line DNA sequence, an additional nucleotide 
sequence was observed between the V H and J H gene seg- 
ments. This nucleotide sequence corresponded to amino 
acids 95 to 97 of the heavy chain. 

From these results, Hood and his colleagues proposed that 
a third germ-line gene segment must join with the V H and J H 
gene segments to encode the entire variable region of the 
heavy chain. This gene segment, which encoded amino acids 
within the third complementarity- determining region 
(CDR3), was designated D for diversity, because of its contri- 
bution to the generation of antibody diversity. Tonegawa and 
his colleagues located the D gene segments within mouse 
germ-line DNA with a cDNA probe complementary to the D 
region, which hybridized with a stretch of DNA lying be- 
tween the V H and J H gene segments. 

The heavy-chain multigene family on human chromo- 
some 14 has been shown by direct sequencing of DNA to 
contain 51 V H gene segments located upstream from a clus- 
ter of 27 functional D H gene segments. As with the light- 
chain genes, each V H gene segment is preceded by a leader 



sequence a short distance upstream. Downstream from the 
D H gene segments are six functional J H gene segments, fol- 
lowed by a series of C H gene segments. Each C H gene seg- 
ment encodes the constant region of an immunoglobulin 
heavy-chain isotype. The C H gene segments consist of coding 
exons and noncoding introns. Each exon encodes a separate 
domain of the heavy-chain constant region. A similar heavy- 
chain gene organization is found in the mouse. 

The conservation of important biological effector func- 
tions of the antibody molecule is maintained by the limited 
number of heavy-chain constant-region genes. In humans 
and mice, the C H gene segments are arranged sequentially in 
the order C^, Cg, C y , C £ , C a (see Figure 5-3c). This sequential 
arrangement is no accident; it is generally related to the se- 
quential expression of the immunoglobulin classes in the 
course of B-cell development and the initial IgM response of 
a B cell to its first encounter with an antigen. 



Variable-Region Gene 
Rearrangements 



The preceding sections have shown that functional genes 
that encode immunoglobulin light and heavy chains are 



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assembled by recombinational events at the DNA level. These 
events and the parallel events involving T-receptor genes are 
the only known site-specific DNA rearrangements in verte- 
brates. Variable-region gene rearrangements occur in an or- 
dered sequence during B-cell maturation in the bone marrow. 
The heavy-chain variable-region genes rearrange first, then 
the light-chain variable-region genes. At the end of this 
process, each B cell contains a single functional variable- 
region DNA sequence for its heavy chain and another for its 
light chain. 

The process of variable-region gene rearrangement pro- 
duces mature, immunocompetent B cells; each such cell is 
committed to produce antibody with a binding site encoded 
by the particular sequence of its rearranged V genes. As de- 
scribed later in this chapter, rearrangements of the heavy- 
chain constant-region genes will generate further changes in 
the immunoglobulin class (isotype) expressed by a B cell, but 
those changes will not affect the cell's antigenic specificity. 

The steps in variable-region gene rearrangement occur in 
an ordered sequence, but they are random events that result 
in the random determination of B-cell specificity. The order, 
mechanism, and consequences of these rearrangements are 
described in this section. 



L V K 1 



LV K 23 LV K » 



Light-Chain DNA Undergoes 
V-J Rearrangements 

Expression of both k and \ light chains requires rearrange- 
ment of the variable-region V and J gene segments. In hu- 
mans, any of the functional V x genes can combine with any 
of the four functional J\-Cx combinations. In the mouse, 
things are slightly more complicated. DNA rearrangement 
can join the V\l gene segment with either the J x l or the J\3 
gene segment, or the V x 2 gene segment can be joined with 
the Jx2 gene segment. In human or mouse k light-chain 
DNA, any one of the V K gene segments can be joined with 
any one of the functional J x gene segments. 

Rearranged k and \ genes contain the following regions in 
order from the 5' to 3' end: a short leader (L) exon, a non- 
coding sequence (intron), a joined VJ gene segment, a second 
intron, and the constant region. Upstream from each leader 
gene segment is a promoter sequence. The rearranged light- 
chain sequence is transcribed by RNA polymerase from the L 
exon through the C segment to the stop signal, generating a 
light-chain primary RNA transcript (Figure 5-4). The in- 
trons in the primary transcript are removed by RNA- 
Qg enzymes, and the resulting light-chain messenger 



J K 



^WW^3< 



V-J joining 



Rearranged K-chain DNA 



UNA transcript 



Nascent poh 



L V K 1 L V K J K J K C K 

-l«--MH]-CIr-3' 

Transcription 
L V K J K J K C K 

Polyadenylation 
RNA splicing 

LVJ C 

— | K | ^Pol 






LVJC K 



I Kappa light-chain gene rearrangement and RNA pro- 
cessing events required to generate a k light-chain protein. In this 
example, rearrangement joins V K 23 and J K 4. 



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Generation of B-Cell and T-Cell Respor 



RNA then exits from the nucleus. The light-chain mRNA 
binds to ribosomes and is translated into the light-chain pro- 
tein. The leader sequence at the amino terminus pulls the 
growing polypeptide chain into the lumen of the rough en- 
doplasmic reticulum and is then cleaved, so it is not present 
in the finished light-chain protein product. 

Heavy-Chain DNA Undergoes 
V-D-J Rearrangements 

Generation of a functional immunoglobulin heavy-chain 
gene requires two separate rearrangement events within the 
variable region. As illustrated in Figure 5-5, a D H gene seg- 
ment first joins to a J H segment; the resulting D H J H segment 
then moves next to and joins a V H segment to generate a 
V H D H J H unit that encodes the entire variable region. In 
heavy-chain DNA, variable-region rearrangement produces 
a rearranged gene consisting of the following sequences, 



starting from the 5' end: a short L exon, an intron, a joined 
VDJ segment, another intron, and a series of C gene seg- 
ments. As with the light-chain genes, a promoter sequence is 
located a short distance upstream from each heavy-chain 
leader sequence. 

Once heavy-chain gene rearrangement is accomplished, 
RNA polymerase can bind to the promoter sequence and 
transcribe the entire heavy-chain gene, including the introns. 
Initially, both C^ and Cg gene segments are transcribed. Dif- 
ferential polyadenylation and RNA splicing remove the in- 
trons and process the primary transcript to generate mRNA 
including either the C^ or the Cg transcript. These two 
iriRNAs are then translated, and the leader peptide of the re- 
sulting nascent polypeptide is cleaved, generating finished jjl 
and 8 chains. The production of two different heavy-chain 
mRNAs allows a mature, immunocompetent B cell to express 
both IgM and IgD with identical antigenic specificity on its 
surface. 



L V H 1 L V H n D H 1 D H 7 D H 13 J H 



C^ C 6 C y 3 C y l C y 2b C y 2a C e 



H]-D-[WHHHH]-C>Oaaaa£>L3h' 



LV H 1 LV H 21 LV H w D H 1D H 6D H J H C,, Cg C y 3 C y l C y 2b C y 2a C E C a 

~ ^h\ Pfoonnonoo^ 



Jr^ 



Rearranged L V„l LV H 20 L V DJ J H C^ C s C y 3 C y l C y 2b C y 2a C E C a 

Transcription 



RNA transcript 


L VDJ C^ Cg 

'4MH}Odr-3' 




L V DJ C^ 

■ 1 1 hCAX 


1 Polyadenylation 

RNA splicing 
i LVDJCj 

« T~r-c*a. 




Translation 




Translation 


polypeptide 


L V DJ q, 


or 


L V DJ C 5 




V DJ C M 


or 


V DJ C 6 




■ II 1 


■ II 1 




[X heavy chain 




8 heavy chain 



ing events required to generate finished u, or 8 heavy-chain protein. involves differential RNA processing, which generates several differ- 

Two DNA joinings are necessary to generate a functional heavy-chain ent products, including |x or 8 heavy chains. Each C gene is drawn as 

gene: a D H to J H joining and a V H to D h Jh joining. In this example, a single coding sequence; in reality, each is organized as a series of 

V H 21 , D H 7, and J H 3 are joined. Expression of functional heavy-chain exons and introns. 



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Mechanism of Variable-Region 
DNA Rearrangements 

Now that we've seen the results of variable-region gene re- 
arrangements, let's examine in detail how this process occurs 
during maturation of B cells. 

Recombination Signal Sequences 
Direct Recombination 

The discovery of two closely related conserved sequences in 
variable-region germ-line DNA paved the way to fuller un- 
derstanding of the mechanism of gene rearrangements. DNA 
sequencing studies revealed the presence of unique recombi- 
nation signal sequences (RSSs) flanking each germ-line V, 
D, and J gene segment. One RSS is located 3' to each V gene 
segment, 5 ' to each J gene segment, and on both sides of each 
D gene segment. These sequences function as signals for the 
recombination process that rearranges the genes. Each RSS 
contains a conserved palindromic heptamer and a conserved 
AT-rich nonamer sequence separated by an intervening se- 
quence of 12 or 23 base pairs (Figure 5-6a). The intervening 
12- and 23-bp sequences correspond, respectively, to one and 
two turns of the DNA helix; for this reason the sequences are 
called one-turn recombination signal sequences and two- 
turn signal sequences. 

The V K signal sequence has a one-turn spacer, and the J K 
signal sequence has a two-turn spacer. In X light-chain DNA, 
this order is reversed; that is, the V x signal sequence has a 
two-turn spacer, and the J x signal sequence has a one-turn 



spacer. In heavy-chain DNA, the signal sequences of the V H 
and J H gene segments have two-turn spacers, the signals on 
either side of the D H gene segment have one-turn spacers 
(Figure 5-6b). Signal sequences having a one-turn spacer can 
join only with sequences having a two-turn spacer (the so- 
called one-turn/two-turn joining rule). This joining rule en- 
sures, for example, that a V L segment joins only to a J L 
segment and not to another V L segment; the rule likewise en- 
sures that V H , D H , and J H segments join in proper order and 
that segments of the same type do not join each other. 

Gene Segments Are Joined by Recombinases 

V-(D)-J recombination, which takes place at the junctions 
between RSSs and coding sequences, is catalyzed by enzymes 
collectively called V(D)J recombinase. 

Identification of the enzymes that catalyze recombination 
of V, D, and J gene segments began in the late 1980s and is still 
ongoing. In 1990 David Schatz, Marjorie Oettinger, and 
David Baltimore first reported the identification of two 
recombination-activating genes, designated RAG- 1 and 
RAG-2, whose encoded proteins act synergistically and are re- 
quired to mediate V-(D)-J joining. The RAG- 1 and RAG-2 pro- 
teins and the enzyme terminal deoxynucleotidyl transferase 
(TdT) are the only lymphoid-specific gene products that 
have been shown to be involved in V-(D)-J rearrangement. 

The recombination of variable-region gene segments 
consists of the following steps, catalyzed by a system of re- 
combinase enzymes (Figure 5-7): 
■ Recognition of recombination signal sequences (RSSs) 

by recombinase enzymes, followed by synapsis in which 



(a) Nucleotide sequence of RSSs 

pl-ACAAA 



CACAGTG-23bp 



GTGTC 
Heptam 



► 

Two-turn RSS 



GGTTTTTGT- 12bp-CACTGTG 



12_bp]-GTGACAC 



(b) Location ol >«lobulin DNA 



X-chain DNA 



-OCIr-3' 



■■CEIh' 



Heavy-chain DNA 5'- 



°H Jh c h 

■■O-OEZh*' 



conserved sequences in light-chain and heavy- 
chain DNA function as recombination signal sequences (RSSs). ter 
(a) Both signal sequences consist of a conserved palindromic hep- lin 
tamer and conserved AT-rich nonamer; these are separated by th< 
nonconserved spacers of 1 2 or 23 base pairs, (b) The two types of tui 



RSS— designated one-turn RSS and two-turn RSS— have char 

teristic locations within X-chain, K-chain, and heavy-chain ger 

ine DNA. During DNA rearrangement, gene segments adjacent 

one-turn RSS can join only with segments adjacent to the tv 

RSS. 



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Generation of B-Cell and T-Cell Respor 



^ RSS 

J, ® Recognition of RSSs J, 

A ■ '• ■ by RAG-1/2 and synapsis 

^3- -■:, ..■■:::■■■■■=*: 



••40=^' 



©Single-strand 
DNA cleavage 
by RAG-1/2 






^^^ 



™ ^~ (3) Hairpin formation ' " ' ^™^ 

and double-strand 
DNA break by 




40 ^ 



■40 ^ 



© Optional addition 
to H-chain segments 
of N-nucleotides by TdT 



::=tO]=* 



o 



^ = One-turn RSS 
[> = Two-turn RSS 



tion of immunoglobulin gene segments is illustrated with V K and J K . 
(a) Deletional joining occurs when the gene segments to be joined 
have the same transcriptional orientation (indicated by horizontal 
blue arrows). This process yields two products: a rearranged VJ unit 
that includes the coding joint, and a circular excision product con- 
sisting of the recombination signal sequences (RSSs), signal joint, 
and intervening DNA. (b) Inversional joining occurs when the gene 
segments have opposite transcriptional orientations. In this case, the 
RSSs, signal joint, and intervening DNA are retained, and the orien- 
tation of one of the joined segments is inverted. In both types of re- 
combination, a few nucleotides may be deleted from or added to the 
cut ends of the coding sequences before they are rejoined. 



two signal sequences and the adjacent coding sequences 
(gene segments) are brought into proximity 

■ Cleavage of one strand of DNA by RAG- 1 and RAG-2 at 
the junctures of the signal sequences and coding sequences 

■ A reaction catalyzed by RAG- 1 and RAG-2 in which the 
free 3' -OH group on the cut DNA strand attacks the 
phosphodiester bond linking the opposite strand to the 
signal sequence, simultaneously producing a hairpin 
structure at the cut end of the coding sequence and a 
flush, 5'-phosphorylated, double-strand break at the 
signal sequence 

■ Cutting of the hairpin to generate sites for the addition 
of P-region nucleotides, followed by the trimming of a 
few nucleotides from the coding sequence by a single- 
strand endonuclease 

■ Addition of up to 15 nucleotides, called N-region 
nucleotides, at the cut ends of the V, D, and J coding 
sequences of the heavy chain by the enzyme terminal 
deoxynucleotidyl transferase 

■ Repair and ligation to join the coding sequences and to 
join the signal sequences, catalyzed by normal double- 
strand break repair (DSBR) enzymes 
Recombination results in the formation of a coding joint, 

falling between the coding sequences, and a signal joint, be- 
tween the RSSs. The transcriptional orientation of the gene 
segments to be joined determines the fate of the signal joint 
and intervening DNA. When the two gene segments are in 
the same transcriptional orientation, joining results in dele- 
tion of the signal joint and intervening DNA as a circular ex- 
cision product (Figure 5-8). Less frequently, the two gene 
segments have opposite orientations. In this case joining oc- 
curs by inversion of the DNA, resulting in 



I 







ular DNA isolated from thymocytes in which the 
DNA encoding the chains of the T-cell receptor (TCR) undergoes re- 
arrangement in a process like that involving the immunoglobulin 
genes. Isolation of this circular excision product is direct evidence for 
the mechanism of deletional joining shown in Figure 5-7. [From K. 
Okazaki et al., 1987, Cell 49:477.] 



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both the coding joint and the signal joint (and ii 
DNA) on the chromosome. In the human k locus, about half 
of the V K gene segments are inverted with respect to J K and 
their joining is thus by inversion. 

Ig-Gene Rearrangements May Be 
Productive or Nonproductive 

One of the striking features of gene-segment recombination 
is the diversity of the coding joints that are formed between 
any two gene segments. Although the double-strand DNA 
breaks that initiate V-(D)-J rearrangements are introduced 
precisely at the junctions of signal sequences and coding se- 
quences, the subsequent joining of the coding sequences is 
imprecise. Junctional diversity at the V-J and V-D-J coding 
joints is generated by a number of mechanisms: ' 
cutting of the hairpin to generate P-nucleotides, a 
trimming of the coding sequences, variation in N-nucleotide 
addition, and flexibility in joining the coding sequences. The 
introduction of randomness in the joining process helps gen- 
erate antibody diversity by contributing to the hypervariabil- 
ity of the antigen-binding site. (This phenomenon is covered 
in more detail below in the section on generation of antibody 
diversity.) 



Another consequence of imprecise joining is that gene 
segments may be joined out of phase, so that the triplet read- 
ing frame for translation is not preserved. In such a nonpro- 
ductive rearrangement, the resulting VJ or VDJ unit is likely 
to contain numerous stop codons, which interrupt transla- 
tion (Figure 5-9). When gene segments are joined in phase, 
the reading frame is maintained. In such a productive re- 
arrangement, the resulting VJ or VDJ unit can be translated 
in its entirety, yielding a complete antibody. 

If one allele rearranges nonproductively, a B cell may still 
be able to rearrange the other allele productively. If an in- 
phase rearranged heavy-chain and light-chain gene are not 
produced, the B cell dies by apoptosis. It is estimated that 
only one in three attempts at V L -J L joining, and one in three 
subsequent attempts at V H -D H J H joining, are productive. As 
a result, less than 1/9 (11%) of the early-stage pre-B cells in 
the bone marrow progress to maturity and leave the bone 
marrow as mature immunocompetent B cells. 

Allelic Exclusion Ensures a Single 
Antigenic Specificity 



B cells, like 

ternal and paternal ch 



diploid and c 

Even though a 




GAGGATGCTCC) |CACAGTG~ 



Productive 
rearrangements 



Nonproductive 
rearrangements 



Glu Asp Ala Thr Arg 

(1) GAGGATGCGACTAGG 

Glu Asp Gly Thr Arg 

(2) GAGGATGGGACTAGG 

Glu Asp Trp Thr Arg 

(3) GAGGATTGGACTAGG 

Glu Asp Ala Asp Stop 
(?) GAGGATGCGGACTAGG 

Glu Val Asp Stop 
(5) GAGGTGGACTAGG 



Junctional flexibility in the joining of 
gene segments is illustrated with V K and J K . In-phas 
1, 2, and 3) generates a productive rearrangemer 
translated into protein. Out-of-phase joining (arrov 
to a nonproductive rearrangement that contains st( 
not translated into protein. 



mmunoglobulir 
: joining (arrow: 
t, which can bi 
s4and 5) lead: 




■ JH I MM I J Because 
heavy- and light-chain ger 
expressed per cell. This pr 
antigenic specificity. The a 
randomly. Thus the expressed ii 



,nly c 



lusion, the immunoglobulin 
le parental chromosome are 
ensures that B cells possess a single 
elected for rearrangement is chosen 
oglobulir 



ternal and one paternal chain or both chains may derive from o 
one parent. Only B cells and T cells exhibit allelic exclusion. Asteri: 
(*) indicate the expressed alleles. 



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Generation of B-Cell and T-Cell Respor 



diploid, it expresses the rearranged heavy-chain genes from 
only one chromosome and the rearranged light-chain genes 
from only one chromosome. The process by which this is ac- 
complished, called allelic exclusion, ensures that functional 
B cells never contain more than one V H D H J H and one V L J L 
unit (Figure 5-10). This is, of course, essential for the 
antigenic specificity of the B cell, because the expression of 
both alleles would render the B cell multispecific. The phe- 
nomenon of allelic exclusion suggests that once a productive 
V H -D H -J H rearrangement and a productive V L -J L rearrange- 
ment have occurred, the recombination machinery is turned 
off, so that the heavy- and light- chain genes on the homolo- 
gous chromosomes are not expressed. 

G. D. Yancopoulos and F. W. Alt have proposed a model to 
account for allelic exclusion (Figure 5-11). They suggest that 
once a productive rearrangement is attained, its encoded 
protein is expressed and the presence of this protein acts as 
a signal to prevent further gene rearrangement. According 
to their model, the presence of jjl heavy chains signals the 



maturing B cell to turn off rearrangement of the other 
heavy- chain allele and to turn on rearrangement of the k 
light-chain genes. If a productive k rearrangement occurs, k 
light chains are produced and then pair with |x heavy chains 
to form a complete antibody molecule. The presence of this 
antibody then turns off further light-chain rearrangement. 
If k rearrangement is nonproductive for both k alleles, re- 
arrangement of the \-chain genes begins. If neither \ allele 
rearranges productively, the B cell presumably ceases to ma- 
ture and soon dies by apoptosis. 

Two studies with transgenic mice have supported the hy- 
pothesis that the protein products encoded by rearranged 
heavy- and light-chain genes regulate rearrangement of the 
remaining alleles. In one study, transgenic mice carrying a 
rearranged u, heavy-chain transgene were prepared. The |x 
transgene product was expressed by a large percentage of the 
B cells, and rearrangement of the endogenous immunoglob- 
ulin heavy-chain genes was blocked. Similarly, cells from a 
transgenic mouse carrying a k light-chain transgene did not 



|i heavy chain inhibits 
rearrangement of |l allele #2 
and induces K rearrangement 



inhibit 

rearrangement of K allele #2 
and X rearrangement 




Cell death 



Model to account for allelic exclusion. Heavy-chain 
genes rearrange first, and once a productive heavy-chain gene 
rearrangement occurs, the jul protein product prevents rearrange- 
ment of the other heavy-chain allele and initiates light-chain gene 

precedes rearrangement of the \ genes, as shown here. In humans, 



either k or \ rearrangement can proceed once a productive heavy- 
chain rearrangement has occurred. Formation of a complete 
immunoglobulin inhibits further light-chain gene rearrangement. If 
a nonproductive rearrangement occurs for one allele, then the eel 
attempts rearrangement of the other allele. [Adapted from C. D. 
Yancopoulos and F. W. Alt, 1986, Annu. Rev. Immunol. 4:339.] 



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' Immunology 5e: 



rearrange the endogenous K-chain genes when the k trans- 
gene was expressed and was associated with a heavy chain to 
form complete immunoglobulin. These studies suggest that 
expression of the heavy- and light-chain proteins may indeed 
prevent gene rearrangement of the remaining alleles and thus 
account for allelic exclusion. 



functional J K> 3 functional J x , and an estimated 13 D H gene 
segments, but only three V x gene segments. Although the 
number of germ-line genes found in either humans or mice 
is far fewer than predicted by early proponents of the germ- 
line model, multiple germ-line V, D, and J gene segments 
clearly do contribute to the diversity of the antigen-binding 
sites in antibodies. 



Generation of Antibody Diversity 

As the organization of the immunoglobulin genes was deci- 
phered, the sources of the vast diversity in the variable region 
began to emerge. The germ-line theory, mentioned earlier, 
argued that the entire variable-region repertoire is encoded 
in the germ line of the organism and is transmitted from par- 
ent to offspring through the germ cells (egg and sperm). The 
somatic-variation theory held that the germ line contains a 
limited number of variable genes, which are diversified in the 
somatic cells by mutational or recombinational events dur- 
ing development of the immune system. With the cloning 
and sequencing of the immunoglobulin genes, both models 
were partly vindicated. 

To date, seven means of antibody diversification have 
been identified in mice and humans: 

■ Multiple germ-line gene segments 

■ Combinatorial V-(D)-J joining 

■ Junctional flexibility 

■ P-region nucleotide addition (P-addition) 

■ N-region nucleotide addition (N-addition) 

■ Somatic hypermutation 

■ Combinatorial association of light and heavy chains 

Although the exact contribution of each of these avenues of 
diversification to total antibody diversity is not known, they 
each contribute significantly to the immense number of dis- 
tinct antibodies that the mammalian immune system is ca- 
pable of generating. 

There Are Numerous Germ-Line 
V, D, and J Gene Segments 

An inventory of functional V, D, and J gene segments in the 
germ-line DNA of one human reveals 51 V H , 25 D, 6 J H , 
40 V K , 5 J K , 31 Vx, and 4 J x gene segments. In addition to these 
functional segments, there are many pseudogenes. It should 
be borne in mind that these numbers were largely derived 
from a landmark study that sequenced the DNA of the 
immunoglobulin loci of a single individual. The immuno- 
globulin loci of other individuals might contain slightly dif- 
ferent numbers of particular types of gene segments. 

In the mouse, although the numbers are known with less 
precision than in the human, there appear to be about 85 V K 
gene segments and 134 V H gene segments, 4 functional J H , 4 



Combinatorial V-J and V-D-J Joining 
Generates Diversity 

The contribution of multiple germ-line gene segments to an- 
tibody diversity is magnified by the random rearrangement 
of these segments in somatic cells. It is possible to calculate 
how much diversity can be achieved by gene rearrangments 
(Table 5-2). In humans, the ability of any of the 51 V H gene 
segments to combine with any of the 27 D H segments and 
any of the 6 J H segments allows a considerable amount of 
heavy-chain gene diversity to be generated (51 X 27 X 6 = 
8262 possible combinations). Similarly, 40 V K gene segments 
randomly combining with 5 J K segments has the potential of 
generating 200 possible combinations at the k locus, while 30 
V x and 4 J x gene segments allow up to 120 possible combina- 
tions at the human \ locus. It is important to realize that 
these are minimal calculations of potential diversity. Junc- 
tional flexibility and P- and N-nucleotide addition, as men- 
tioned above, and, especially, somatic hypermutation, which 
will be described shortly, together make an enormous contri- 
bution to antibody diversity. Although it is not possible to 
make an exact calculation of their contribution, most work- 
ers in this field agree that they raise the potential for antibody 
combining-site diversity in humans to well over 10 10 . This 
does not mean that, at any given time, a single individual has 
a repertoire of 10 10 different antibody combining sites. These 
very large numbers describe the set of possible variations, of 
which any individual carries a subset that is smaller by several 



orders of rr 






Junctional Flexibility Adds Diversity 

The enormous diversity generated by means of V, D, and J 
combinations is further augmented by a phenomenon called 
junctional flexibility. As described above, recombination in- 
volves both the joining of recombination signal sequences to 
form a signal joint and the joining of coding sequences to 
form a coding joint (see Figure 5-7). Although the signal se- 
quences are always joined precisely, joining of the coding se- 
quences is often imprecise. In one study, for example, joining 
of the V K 21 and J K 1 coding sequences was analyzed in several 
pre-B cell lines. Sequence analysis of the signal and coding 
joints revealed the contrast in junctional precision (Figure 
5-12). 

As illustrated previously, junctional flexibility leads to 
many nonproductive rearrangements, but it also generates 
productive combinations that encode alternative amino 
acids at each coding joint (see Figure 5-9), thereby increasing 
antibody diversity. The amino acid sequence variation gener- 



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Generation of B-Cell and T-Cell Respor 



tmmmm 



Multiple germ-line segments 



LIGHT CHAINS 



ESTIMATED NUMBER OF SEGMENTS If 



Combinatorial V-D-J and V-J joining 
(possible number of combinations) 

Possible combinatorial associations of 
heavy and light chains ' 



51 X 27 X 6 = 8262 



40 X 5 = 200 



8262 X (200 X 120) = 2.64 X 10 6 



30 X 4 = 120 



ESTIMATED NUMBER OF SEGMENTS IN MICE* 



134 X 13 X 4 = 6968 



] 

Combinatorial V-D-J and V-J joining 

(possible number of combinations) 
Possible combinatorial associations 

of heavy and light chains' 



functional gene segments have been listed. The genome contains additional segmer 
mouse case, the figures contained in the table are only best estimates, because the I 

because of the diversity contributed by junctional flexibility, P-region nucleotide add 



85 X 4 = 340 



< (340 + 6) = 2.41 X 10 6 



mpletely sequenced. 



5'...CACTGTG GTGGAC GTT . . . 3' 
V K 21 
5'...GGATCCTCCC |( 



Coding joints 
(V K 21J K 1) 



5' GGATCC GGACGTT -3' 5' CACTGTG Q 



5'-CACTGTG C 



Cell line #2 5'-GGATC TGGACGTT -3' 



Cell line #3 5'-GGATCCTC GTG 



Cell line #4 5'-GGATCCT TGGACGTT-3' 5'-CACTGTGG 



e for junctional flexibility in im- sequence constancy in the signal joints contrasts with the sequenc 

:. The nucleotide sequences flank- variability in the coding joints. Pink and yellow shading indicate ni 

ing the coding joints between V K 21 and J K 1 and the corresponding cleotides derived from V K 21 and J K 1, respectively, and purple and o 

signal joint sequences were determined in four pre-B cell lines. The ange shading indicate nucleotides from the two RSSs. 



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ated by junctional flexibility in the coding joints has been 
shown to fall within the third hypervariable region (CDR3) 
in immunoglobulin heavy-chain and light-chain DNA 
(Table 5-3). Since CDR3 often makes a major contribution to 
antigen binding by the antibody molecule, amino acid 
changes generated by junctional flexibility are important in 
the generation of antibody diversity. 

P-Addition Adds Diversity 
at Palindromic Sequences 

As described earlier, after the initial single-strand DNA cleav- 
age at the junction of a variable-region gene segment and at- 
tached signal sequence, the nucleotides at the end of the 
coding sequence turn back to form a hairpin structure (see 
Figure 5-7). This hairpin is later cleaved by an endonuclease. 
This second cleavage sometimes occurs at a position that 
leaves a short single strand at the end of the coding sequence. 
The subsequent addition of complementary nucleotides to 
this strand (P-addition) by repair enzymes generates a palin- 
dromic sequence in the coding joint, and so these nucleotides 
are called P-nucleotides (Figure 5- 13a). Variation in the po- 
sition at which the hairpin is cut thus leads to variation in the 
sequence of the coding joint. 

N-Addition Adds Considerable Diversity 
by Addition of Nucleotides 

Variable-region coding joints in rearranged heavy-chain 
genes have been shown to contain short amino acid se- 
quences that are not encoded by the germ-line V, D, or J gene 
segments. These amino acids are encoded by nucleotides 
added during the D-J and V to D-J joining process by a ter- 
minal deoxynucleotidyl transferase (TdT) catalyzed reaction 




encoded by: 



Somatic 



V segment V segment 



(Figure 5- 13b). Evidence that TdT i 
dition of these N-nucleotides has 
studies in fibroblasts. When fibrobla 
the RAG-1 and RAG-2 genes, V-D-J 
but no N-nucleotides were present ii 



; responsible for the ad- 
:ome from transfection 
its were transfected with 



ever, when the fibroblasts 
encoding TdT, then V-D-J re, 
by addition of N-nucleotides 
Up to 15 N-nucleotides ci 
and V H -D H J H joints. Thus, a com 
region is encoded by a V H ND H NJ H 
chain diversity generated by 



the coding joints. How- 



also transfected with the 



rangement was accompanied 
the coding joints, 
be added to both the D H -J H 
)mplete heavy-chain variable 
unit. The additional heavy - 
sl-region nucleotide 
addition is quite large because N regions appear to consist of 
wholly random sequences. Since this diversity occurs at V-D-J 
coding joints, it is localized in CDR3 of the heavy-chain genes. 

Somatic Hypermutation Adds Diversity 
in Already-Rearranged Gene Segments 

All the antibody diversity described so far stems from mech- 
anisms that operate during formation of specific variable 
regions by gene rearrangement. Additional antibody diver- 
sity is generated in rearranged variable-region gene units by 
a process called somatic hypermutation. As a result of so- 
matic hypermutation, individual nucleotides in VJ or VDJ 
units are replaced with alternatives, thus potentially altering 
the specificity of the encoded immunoglobulins. 

Normally, somatic hypermutation occurs only within 
germinal centers (see Chapter 1 1 ), structures that form in sec- 
ondary lymphoid organs within a week or so of immuniza- 
tion with an antigen that activates a T-cell-dependent B-cell 
response. Somatic hypermutation is targeted to rearranged V- 
regions located within a DNA sequence containing about 
1500 nucleotides, which includes the whole of the VJ or VDJ 
segment. Somatic hypermutation occurs at a frequency ap- 
proaching 10~ 3 per base pair per generation. This rate is at 
least a hundred thousand-fold higher (hence the name hyper- 
mutation) than the spontaneous mutation rate, about 
10~ 8 /bp/generation, in other genes. Since the combined 
length of the H-chain and L-chain variable-region genes is 
about 600 bp, one expects that somatic hypermutation will 
introduce at least one mutation per every two cell divisions in 
the pair of V H and V L genes that encode an antibody. 

The mechanism of somatic hypermutation has not yet been 
determined. Most of the mutations are nucleotide substitutions 
rather than deletions or insertions. Somatic hypermutation in- 
troduces these substitutions in a largely, but not completely, 
random fashion. Recent evidence suggests that certain nu- 
cleotide motifs and palindromic sequences within V H and V L 
may be especially susceptible to somatic hypermutation. 

Somatic hypermutations occur throughout the VJ or VDJ 
segment, but in mature B cells they are clustered within the 
CDRs of the V H and V L sequences, where they are most likely 
to influence the overall affinity for antigen. Following expo- 
sure to antigen, those B cells with higher- affinity receptors 
will be preferentially selected for survival. This result of this 



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Generation of B-Cell and T-Cell Respor 



(b) N-nucleotide additi 



=@I&> ci$[7]= 



=^Ic c P cl$[7]= 



^ T ' 



tLTj^ 











1 


Repair enzyme 














complementary nucleotides 








— L^Jagct atat LLI — 

P-nucleotide and N-nucleotide 
eavage of the hairpin intermedial 


additio 
yields 










i du 


join 


ng. (a) 


Ifc 


dou 


doe 


ded e 


nd o 


n the 


oding seque 


ice, then P-nu 
wever, cleavag 


cleotide 


add 


sirs 


ided 


end 


Dur 


ng subseq 


ent repair, 


comple 


men 


nuc 


eotide. 


an 


adde 


d, called P-r 


ucleotides, tc 


produ 


;e p 



^ T< 



TLlJ^ 



I TdT adds N-nucleotides 

Repair enzymes add 
T- complementary nucleotides 



dromic sequences (indicated by brackets). In this example, four 
extra base pairs (blue) are present in the coding joint as the result 
of P-nucleotide addition, (b) Besides P-nucleotide addition, addi- 
tion of random N-nucleotides (light red) by a terminal deoxynu- 
cleotidyl transferase (TdT) can occur during joining of heavy-chain 
coding sequences. 



differential selection is an increase in the antigen affinity of a 
population of B cells. The overall process, called affinity 
maturation, takes place within germinal centers, and is de- 
scribed more fully in Chapter 11. 

Claudia Berek and Cesar Milstein obtained experimental 
evidence demonstrating somatic hypermutation during the 
course of an immune response to a hapten-carrier conju- 
gate. These researchers were able to sequence mRNA that 
encoded antibodies raised against a hapten in response to 
primary, secondary, or tertiary immunization (first, second, 
or third exposure) with a hapten-carrier conjugate. The 
hapten they chose was 2-phenyl-5-oxazolone (phOx), cou- 
pled to a protein carrier. They chose this hapten because it 
had previously been shown that the majority of antibodies 
it induced were encoded by a single germ-line V H and V K 
gene segment. Berek and Milstein immunized mice with the 
phOx-carrier conjugate and then used the mouse spleen 
cells to prepare hybridomas secreting monoclonal anti- 
bodies specific for the phOx hapten. The mRNA sequence 
for the H chain and k light chain of each hybridoma was 
then determined to identify deviations from the germ-line 
sequences. 

The results of this experiment are depicted in Figure 
5-14. Of the 12 hybridomas obtained from mice seven days 
after a primary immunization, all used a particular V H , the 
V H Ox-1 gene segment, and all but one used the same V L 
gene segment, V K Ox-1. Moreover, only a few mutations 
from the germ-line sequence were present in these hybrido- 
mas. By day 14 after primary immunization, analysis of eight 
hybridomas revealed that six continued to use the germ-line 
V H Ox- 1 gene segment and all continued to use the V K Ox- 1 
gene segment. Now, however, all of these hybridomas 



included one or more mutations from the germ-line 
sequence. Hybridomas analyzed from the secondary and 
tertiary responses showed a larger percentage utilizing 
germ-line V H gene segments other than the V H Ox-1 gene. 
In those hybridoma clones that utilized the V H Ox- 1 and V K 
Ox-1 gene segments, most of the mutations were clustered 
in the CDR1 and CDR2 hypervariable regions. The number 
of mutations in the anti-phOx hybridomas progressively in- 
creased following primary, secondary, and tertiary immu- 
nizations, as did the overall affinity of the antibodies for 
phOx (see Figure 5-14). 

A Final Source of Diversity Is Combinatorial 
Association of Heavy and Light Chains 

In humans, there is the potential to generate 8262 heavy- 
chain genes and 320 light-chain genes as a result of variable- 
region gene rearrangements. Assuming that any one of the 
possible heavy-chain and light-chain genes can occur ran- 
domly in the same cell, the potential number of heavy- and 
light-chain combinations is 2,644,240. This number is prob- 
ably higher than the amount of combinatorial diversity actu- 
ally generated in an individual, because it is not likely that all 
V H and V L will pair with each other. Furthermore, the re- 
combination process is not completely random; not all V H , 
D, or V L gene segments are used at the same frequency. Some 
are used often, others only occasionally, and still others al- 
most never. 

Although the number of different antibody combining 
sites the immune system can generate is difficult to calculate 
with precision, we know that it is quite high. Because the 
very large number of new sequences created by junctional 



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u 



Heavy-chain V regioi 



=t=fe 



CDR1 CDR2 



+- 



=3=fc 



sof 



mRNAsequ< 
mas specific for the phOx hapte 
sent the germ-line V H and V K Ox- 
sequences derived from other ge 
the areas where mutations clus 



Experimental evidence for somati 
mmunoglobulin genes. The diagi 
if heavy chains and of light chi 



sof 



cid than the ger 






compares the 
from hybrido- 
i. The horizontal solid lines repre- 
sequences; dashed lines represent 
m-line genes. Blue shading shows 
;red; the blue circles with vertical 
that encode a different amino 
These data show that the fre- 



quency of mutation (1) increases in the course of the primary re- 
sponse (day 7 vs. day 14) and (2) is higher after secondary and ter- 
tiary immunizations than after primary immunization. Moreover, the 
dissociation constant (K d ) of the anti-phOx antibodies decreases dur- 
ing the transition from the primary to tertiary response, indicating an 
increase in the overall affinity of the antibody. Note also that most of 
the mutations are clustered within CDR1 and CDR2 of both the heavy 
and the light chains. [Adapted from C. Berek and C. Milste'm, 1987, Im- 
munol. Rev. 96:23.] 



flexibility, P-nucleotide addition, and N-nucleotide addition 
are within the third CDR, they are positioned to influence 
the structure of the antibody binding site. In addition to 
these sources of antibody diversity, the phenomenon of so- 
matic hypermutation contributes enormously to the reper- 
toire after antigen stimulation. 



Class Switching among 
Constant-Region Genes 

After antigenic stimulation of a B cell, the heavy-chain DNA 
can undergo a further rearrangement in which the V H D H J H 



unit can combine with any C H gene segment. The exact 
mechanism of this process, called class switching or iso- 
type switching, is unclear, but it involves DNA flanking 
sequences (called switch regions) located 2-3 kb upstream 
from each C H segment (except C 8 ). These switch regions, 
though rather large (2 to 10 kb), are composed of multiple 
copies of short repeats (GAGCT and TGGGG). One hy- 
pothesis is that a protein or system of proteins that consti- 
tute the switch recombinase recognize these repeats and 
upon binding carry out the DNA recombination that results 
in class switching. Intercellular regulatory proteins known 
as cytokines act as "switch factors" and play major roles in 
determining the particular immunoglobulin class that is ex- 
pressed as a consequence of switching. Interleukin 4 (IL-4), 



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Generation of B-Cell and T-Cell Respor 



L V DJ C^ C 6 C y 3 C y l C y 2b C y 2a 

S y 3 S T 1 ' S T 2b ' ' S T 2a 




I Proposed mechanism for class switching induced Identification of the indicated circular excision products contai 

by interleukin 4 in rearranged immunoglobulin heavy-chain genes. A portions of the switch sites suggested that IL-4 induces seque 
switch site is located upstream from each C H segment except C s . class switching from C^ to C 7 1 to C e . 



for example, induces class switching from C^ to C 7 1 or C E . 
In some cases, IL-4 has been observed to induce class switch- 
ing in a successive manner: first from C^ to C 7 1 and then 
from Cyl to C e (Figure 5-15). Examination of the DNA ex- 
cision products produced during class switching from C^, to 
C 7 1 showed that a circular excision product containing C^ 
together with the 5' end of the 7I switch region (S 7 1) and 
the 3' end of the |x switch region (S^) was generated. Fur- 
thermore, the switch from C 7 1 to C e produced circular exci- 
sion products containing C 7 1 together with portions of the 
(jl, 7, and e switch regions. Thus class switching depends 
upon the interplay of three elements: switch regions, a 
switch recombinase, and the cytokine signals that dictate the 
isotype to which the B cell switches. A more complete de- 



scription of the role of cytokines ii 
in Chapter 11. 



Expression of Ig Genes 

As in the expression of other genes, post-transcriptional 
processing of immunoglobulin primary transcripts is 
required to produce functional mRNAs (see Figures 5-4 
and 5-5). The primary transcripts produced from re- 
arranged heavy-chain and light-chain genes contain inter- 
vening DNA sequences that include noncoding introns and 
J gene segments not lost during V-(D)-J rearrangement. 
In addition, as noted earlier, the heavy-chain C-gene 



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' Immunology 5e: 



segments are organized as a series of coding exons and 
noncoding introns. Each exon of a C H gene segment corre- 
sponds to a constant-region domain or a hinge region of 
the heavy-chain polypeptide. The primary transcript must 
be processed to remove the intervening DNA sequences, 
and the remaining exons must be connected by a process 
called RNA splicing. Short, moderately conserved splice 
sequences, or splice sites, which are located at the intron- 
exon boundaries within a primary transcript, signal the 
positions at which splicing occurs. Processing of the pri- 
mary transcript in the nucleus removes each of these in- 
tervening sequences to yield the final mRNA product. The 
mRNA is then exported from the nucleus to be translated 
by ribosomes into complete H or L chains. 



Heavy-Chain Primary Transcripts Undergo 
Differential RNA Processing 

Processing of an immunoglobulin heavy-chain primary 
transcript can yield several different mRNAs, which explains 
how a single B cell can produce secreted or membrane- 
bound forms of a particular immunoglobulin and simulta- 
neously express IgM and IgD. 



EXPRESSION OF MEMBRANE OR SECRETED 
IMMUNOGLOBULIN 

As explained in Chapter 4, a particular immunoglobulin can 
exist in either membrane-bound or secreted form. The two 
forms differ in the amino acid sequence of the heavy-chain 
carboxyl-terminal domains (C H 3/C H 3 in IgA, IgD, and IgG 
and C H 4/C H 4 in IgE and IgM). The secreted form has a hy- 
drophilic sequence of about 20 amino acids in the carboxyl- 
terminal domain; this is replaced in the membrane-bound 
form with a sequence of about 40 amino acids containing a 
hydrophilic segment that extends outside the cell, a hy- 
drophobic transmembrane segment, and a short hydrophilic 
segment at the carboxyl terminus that extends into the cyto- 
plasm (Figure 5-16a). For some time, the existence of these 
two forms seemed inconsistent with the structure of germ- 
line heavy-chain DNA, which had been shown to contain a 
single C H gene segment corresponding to each class and 
subclass. 

The resolution of this puzzle came from DNA sequenc- 
ing of the C^ gene segment, which consists of four exons 
(C^l, C^2, C^3, and 0^4) corresponding to the four do- 
mains of the IgM molecule. The 0^4 exon contains a nu- 
cleotide sequence (called S) at its 3' end that encodes the 
hydrophilic sequence in the C H 4 domain of secreted IgM. 
Two additional exons called Ml and M2 are located just 
1.8 kb downstream from the 3' end of the C^4 exon. The 
Ml exon encodes the transmembrane segment, and M2 
encodes the cytoplasmic segment of the C H 4 domain in 
membrane-bound IgM. Later DNA sequencing revealed 



that all the C H gene segments have two additional down- 
stream Ml and M2 exons that encode the transmembrane 
and cytoplasmic segments. 

The primary transcript produced by transcription of a re- 
arranged jjl heavy-chain gene contains two polyadenylation 
signal sequences, or poly-A sites, in the C^ segment. Site 1 is 
located at the 3' end of the 0^4 exon, and site 2 is at the 3' 
end of the M2 exon (Figure 5- 16b). If cleavage of the pri- 
mary transcript and addition of the poly-A tail occurs at site 
1, the Ml and M2 exons are lost. Excision of the introns and 
splicing of the remaining exons then produces mRNA en- 
coding the secreted form of the heavy chain. If cleavage and 
polyadenylation of the primary transcript occurs instead at 
site 2, then a different pattern of splicing results. In this case, 
splicing removes the S sequence at the 3' end of the C^4 
exon, which encodes the hydrophilic carboxyl-terminal end 
of the secreted form, and joins the remainder of the C^4 
exon with the Ml and M2 exons, producing mRNA for the 
membrane form of the heavy chain. 

Thus, differential processing of a common primary tran- 
script determines whether the secreted or membrane form 
of an immunoglobulin will be produced. As noted previ- 
ously, mature naive B cells produce only membrane-bound 
antibody, whereas differentiated plasma cells produce se- 
creted antibodies. It remains to be determined precisely how 
naive B cells and plasma cells direct RNA processing prefer- 
entially toward the production of mRNA encoding one form 
or the other. 



SIMULTANEOUS EXPRESSION OF IgM AND IgD 
Differential RNA processing also underlies the simultane- 
ous expression of membrane-bound IgM and IgD by ma- 
ture B cells. As mentioned already, transcription of 
rearranged heavy-chain genes in mature B cells produces 
primary transcripts containing both the C^ and C s gene 
segments. The C^ and C 8 , gene segments are close together 
in the rearranged gene (only about 5 kb apart), and the lack 
of a switch site between them permits the entire VDJC^Cs 
region to be transcribed into a single primary RNA tran- 
script about 15 kb long, which contains four poly-A sites 
(Figure 5-17a). Sites 1 and 2 are associated with C^, as de- 
scribed in the previous section; sites 3 and 4 are located at 
similar places in the C s gene segment. If the heavy-chain 
transcript is cleaved and polyadenylated at site 2 after the 
C^ exons, then the mRNA will encode the membrane form 
of the (JL heavy chain (Figure 5-17b); if polyadenylation is 
instead further downstream at site 4, after the C s exons, 
then RNA splicing will remove the intervening C^, exons 
and produce mRNA encoding the membrane form of the 8 
heavy chain (Figure 5-17c). 

Since the mature B cell expresses both IgM and IgD on 
its membrane, both processing pathways must occur si- 
multaneously. Likewise, cleavage and polyadenylation of 
the primary heavy-chain transcript at poly-A site 1 or 3 in 



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Generation of B-Cell and T-Cell Respor 




Hydrophilic 
| | Hydrophobic 



Encoded by 
Ml andM2 < 
exons of C„ Membrane 





C (l 4 


















-(e) 


->68 




® 

- (e) 568 






Wl1_ 






JiMJ^m) 


576- 






594- 




(Fp94 




+ (K) 



L Cytoplasm COOH 












Primary L , ^ J H 1 V 2 ^ ^ s M1 M2 

transcript \ \ \ \ 

Poly-A Poly-A Poly-A Poly-A 




RNA transcript for secreted \l 
L VDJ J |ll Jl2 u3 u4S 



RNA transcript for men 
L V DJ J ul u2 u3 u4 S Ml M2 



■ipi£^pqp-<«. pi^pppfw 



L V DJ ul u2 u3 u4 S 
I III I I I h^ 
mRNA encoding secreted u chain 



L V DJ ul u2 u3 U4M1M2 
■ II I I I ■ 
mRNA encoding membrane u chain 



the heavy chain by alternative RNA processing, (a) Amino acid mary transcript of a rearranged heavy-chain gene showing the C^ 

sequence ofthe carboxyl-terminal end of secreted and membrane exons and poly-A sites. Polyadenylation of the primary transcript 

jjl heavy chains. Residues are indicated by the single-letter amino at either site 1 or site 2 and subsequent splicing (indicated by V- 

acid code. Hydrophilic and hydrophobic residues and regions are shaped lines) generates mRNAs encoding either secreted or 

indicated by purple and orange, respectively, and charged amino membrane jul chains, 
acids are indicated with a + or -. The white regions of the 



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Organization and Expression of Immunoglobulin Genes chapter 5 125 



(a) H-chain primary transcript 

C„ C 8 

VDJ ' A ■> ' A "> 

L^^^^ J u.1 |i2 u3 u4S Ml M2 81 52 83 S M1M2 

^'-ltMKXXX> — HKXX3 , ■ i *' 

\ / \ \ \ \ 

-6.5 Poly-A Poly-A Poly-A Poly-A 



(b) Polyadenylation of primary transcript at 



S Ml M2 



Uplicing 




Uplicing 

VDJ 
L^1^8l 82 83M1M2 

- Ml I I ft ^ 



' Expression of membrane forms of |ul and 8 heavy adenylation at site 2 and splicing, (c) Structure of 8 m transcript and 

chains by alternative RNA processing, (a) Structure of rearranged § m mRNA resulting from polyadenylation at site 4 and splicing, 

heavy-chain gene showing C^ and C 8 exons and poly-A sites, (b) Both processing pathways can proceed in any given B cell. 
Structure of |x m transcript and |X m mRNA resulting from poly- 



plasma cells and subsequent splicing will yield the secreted 
form of the (jl or 8 heavy chains, respectively (see Figure 
5-16b). 



Synthesis, Assembly, and Secretion of 
Immunoglobulins 

Immunoglobulin heavy- and light-chain mRNAs are 
translated on separate polyribosomes of the rough endo- 
plasmic reticulum (RER). Newly synthesized chains con- 
tain an amino-terminal leader sequence, which serves to 
guide the chains into the lumen of the RER, where the sig- 
nal sequence is then cleaved. The assembly of light (L) and 
heavy (H) chains into the disulfide-linked and glycosylated 
immunoglobulin molecule occurs as the chains pass 
through the cisternae of the RER. The complete molecules 
are transported to the Golgi apparatus and then into 



secretory vesicles, which fuse with the plasma membrane 
(Figure 5-18). 

The order of chain assembly varies among the im- 
munoglobulin classes. In the case of IgM, the H and L chains 
assemble within the RER to form half-molecules, and then 
two half-molecules assemble to form the complete molecule. 
In the case of IgG, two H chains assemble, then an H 2 L inter- 
mediate is assembled, and finally the complete H 2 L 2 mole- 
cule is formed. Interchain disulfide bonds are formed, and 
the polypeptides are glycosylated as they move through the 
Golgi apparatus. 

If the molecule contains the transmembrane sequence of 
the membrane form, it becomes anchored in the membrane 
of a secretory vesicle and is inserted into the plasma mem- 
brane as the vesicle fuses with the plasma membrane (see 
Figure 5-18, insert). If the molecule contains the hydrophilic 
sequence of secreted immunoglobulins, it is transported as a 
free molecule in a secretory vesicle and is released from the 
cell when the vesicle fuses with the plasma membrane. 



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Generation of B-Cell and T-Cell Respor 




BS5S5" 



Light-chain Nascent Heavy-chain 

translation Ig (leader translation 



munoglobulin molecule. The heavy and light chains are synthesized 
on separate polyribosomes (polysomes). The assembly of the 
chains to form the disulfide-linked immunoglobulin molecule oc- 
curs as the chains pass through the cisternae of the rough endo- 
plasmic reticulum (RER) into the Golgi apparatus and then into 
secretory vesicles. The main figure depicts assembly of a secreted 
antibody. The inset depicts a membrane-bound antibody, which 
contains the carboxyl-terminal transmembrane segment. This form 
becomes anchored in the membrane of secretory vesicles and then 
is inserted into the cell membrane when the vesicles fuse with the 



Regulation of Ig-Gene Transcription 

The immunoglobulin genes are expressed only in B -lineage 
cells, and even within this lineage, the genes are expressed at 
different rates during different developmental stages. As with 
other eukaryotic genes, three major classes of cis regulatory 
sequences in DNA regulate transcription of immunoglobu- 
lin genes: 

■ Promoters: relatively short nucleotide sequences, 
extending about 200 bp upstream from the transcription 
initiation site, that promote initiation of RNA 
transcription in a specific direction 

■ Enhancers: nucleotide sequences situated some distance 
upstream or downstream from a gene that activate 
transcription from the promoter sequence in an 
orientation-independer 



■ Silencers: nucleotide sequences that down-regulate 
transcription, operating in both directions over a 
distance. 

The locations of the three types of regulatory elements in 
germ-line immunoglobulin DNA are shown in Figure 5-19. 
All of these regulatory elements have clusters of sequence 
motifs that can bind specifically to one or more nuclear pro- 
Each V H and V L gene segment has a promoter located just 
upstream from the leader sequence. In addition, the Jk 
cluster and each of the D H genes of the heavy-chain locus 
are preceded by promoters. Like other promoters, the 
immunoglobulin promoters contain a highly conserved AT- 
rich sequence called the TATA box, which serves as a site for 
the binding of a number of proteins that are necessary for the 
initiation of RNA transcription. The actual process of tran- 
scription is performed by RNA polymerase II, which starts 
transcribing DNA from the initiation site, located about 25 
bp downstream of the TATA box. Ig promoters also contain 
an essential and conserved octamer that confers B-cell speci- 
ficity on the promoter. The octamer binds two transcription 
factors, oct-1, found in many cell types, and oct-2, found 
only in B cells. 

While much remains to be learned about the function of 
enhancers, they have binding sites for a number of proteins, 
many of which are transcription factors. A particularly im- 
portant role is played by two proteins encoded by the E2A 
gene which can undergo alternate splicing to generate two 
collaborating proteins, both of which bind to the u, and k in- 
tronic enhancers. These proteins are essential for B-cell de- 
velopment and E2A knockout mice make normal numbers of 
T cells but show a total absence of B cells. Interestingly, trans- 
fection of these enhancer-binding proteins into a T cell line 
resulted in a dramatic increase in the transcription of u, chain 
mRNA and even induced the T cell to undergo D H + Jh -» 
D H J H rearrangement. Silencers may inhibit the activity of Ig 



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Organization and Expression of Immunoglobulin Genes chapter 5 12} 

H-chain DNA 

p L V H p L V H D H J H E C^ C g C y 3 C y l C y 2b C y 2a C e C a 3' E 



K-chain DNA 

P L V K P L V K P L V K J K Ek C K 3 ' k e 

^^ Silencers ^ 



Key = Promoter £ 
Enhancer ^fc 



X-chain DNA 

P L V X 2 J X 2C X 2 J X 4 Q4 ^ 2 _ 4E P LV^l J^3 Cjg J x l C x l &3-1E 

^-•-hEh-O-EhB-ED • // • ■ D^H]-CIH]-CIb^»- 



3' 



(green), and silencers (yellow) in mouse heavy-chain, k light-chain, that precede the DH cluster, a number of the C genes and the J x clus 
and X light-chain germ-line DNA. Variable-region DNA rearrange- ter are omitted from this diagram for the sake of clarity, 
ment moves an enhancer close enough to a promoter that the en- 



enhancers in non-B cells. If so, they could be important con- 
tributors to the high levels of Ig gene transcription that are 
characteristic of B cells but absent in other cell types. 

One heavy-chain enhancer is located within the intron 
between the last (3') J gene segment and the first (5') C gene 
segment (C^), which encodes the |x heavy chain. Because 
this heavy-chain enhancer (EjJ is located 5' of the S^. switch 
site near C^, it can continue to function after class switching 
has occurred. Another heavy-chain enhancer (3' a E) has 
been detected 3' of the C a gene segment. One k light-chain 
enhancer (E K ) is located between the J K segment and the 
C K segment, and another enhancer (3' K E) is located 3' of 
the C K segment. The X. light-chain enhancers are located 3' 
of C^4 and 3' of Cxi- Silencers have been identified in 
heavy-chain and K-chain DNA, adjacent to enhancers, but 
not in A-chain DNA. 

DNA Rearrangement Greatly 
Accelerates Transcription 

The promoters associated with the immunoglobulin V gene 
segments bind RNA polymerase II very weakly, and the vari- 
able-region enhancers in germ-line DNA are quite distant 
from the promoters (about 250-300 kb), too remote to signif- 
icantly influence transcription. For this reason, the rate of 
transcription of V H and V L coding regions is negligible in un- 
rearranged germ-line DNA. Variable-region gene rearrange- 
ment brings a promoter and enhancer within 2 kb of each 
other, close enough for the enhancer to influence transcription 
from the nearby promoter. As a result, the rate of transcription 
of a rearranged V L J L or V H D H J H unit is as much as 10 4 times 
the rate of transcription of unrearranged V L or V H segments. 
This effect was demonstrated directly in a study in which B 



cells transfected with rearranged heavy-chain genes from 
which the enhancer had been deleted did not transcribe the 
genes, whereas B cells transfected with similar genes that con- 
tained the enhancer transcribed the transfected genes at a high 
rate. These findings highlight the importance of enhancers in 
the normal transcription of immunoglobulin genes. 

Genes that regulate cellular proliferation or prohibit cell 
death sometimes translocate to the immunoglobulin heavy- 
or light-chain loci. Here, under the influence of an im- 
munoglobulin enhancer, the expression of these genes is sig- 
nificantly elevated, resulting in high levels of growth 
promoting or cell death inhibiting proteins. Translocations 
of the c-myc and bcl-2 oncogenes have each been associated 
with malignant B-cell lymphomas. The translocation of c- 
myc leads to constitutive expression of c-Myc and an aggres- 
sive, highly proliferative B-cell lymphoma called Burkitt's 
lymphoma. The translocation of bcl-2 leads to suspension of 
programmed cell death in B cells, resulting in follicular B-cell 
lymphoma. These cancer-promoting translocations are cov- 
ered in greater detail in Chapter 22. 

Ig-Gene Expression Is Inhibited in T Cells 

As noted earlier, germ-line DNA encoding the T-cell receptor 
(TCR) undergoes V-(D)-J rearrangement to generate func- 
tional TCR genes. Rearrangement of both immunoglobulin 
and TCR germ-line DNA occurs by similar recombination 
processes mediated by RAG-1 and RAG-2 and involving re- 
combination signal sequences with one-turn or two-turn 
spacers (see Figure 5-7). Despite the similarity of the 
processes, complete Ig-gene rearrangement of H and L 
chains occurs only in B cells and complete TCR-gene 
rearrangement is limited to T cells. 



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Hitoshi Sakano and coworkers have obtained results sug- 
gesting that a sequence within the K-chain 3' enhancer (3' K E) 
serves to regulate the joining of V K to J K in B and T cells. 
When a sequence known as the PU.l binding site within the 
3' K-chain enhancer was mutated, these researchers found 
that V K -J K joining occurred in T cells as well as B cells. They 
propose that binding of a protein expressed by T cells, but 
not B cells, to the unmutated K-chain enhancer normally 
prevents V K -J K joining in T cells. The identity of this DNA- 
binding protein in T cells remains to be determined. Similar 
processes may prevent rearrangement of heavy-chain and X.- 
chain DNA in T cells. 



Antibody Genes and Antibody 
Engineering 

There are many clinical applications in which the exquisite 
specificity of a mouse monoclonal antibody would be useful. 
However, when mouse monoclonal antibodies are intro- 
duced into humans they are recognized as foreign and evoke 
an antibody response that quickly clears the mouse mono- 
clonal antibody from the bloodstream. In addition, circulat- 
ing complexes of mouse and human antibodies can cause 
allergic reactions. In some cases, the buildup of these com- 
plexes in organs such as the kidney can cause serious and 
even life-threatening reactions. Clearly, one way to avoid 
these undesirable reactions is to use human monoclonal a 
tibodies for clinical applications. However, the preparation 
human monoclonal antibodies has been hampered by n 
merous technical problems. In response to the difficulty 
producing human monoclonal antibodies and the complica- 
tions resulting from the use of mouse monoclonal antibodies 
in humans, there is now a major effort to engineer mono- 
clonal antibodies and antibody binding sites with recombi- 
nant DNA technology. 

The growing knowledge of antibody gene structure and 
regulation has made possible what Cesar Milstein, one of the 
inventors of monoclonal antibody technology, has called 
"man-made antibodies." It is now possible to design and con- 
struct genes that encode immunoglobulin molecules in 
which the variable regions come from one species and the 
constant regions come from another. New genes have been 
created that link nucleotide sequences coding nonantibody 
proteins with sequences that encode antibody variable re- 
gions specific for particular antigens. These molecular hy- 
brids or chimeras may be able to deliver powerful toxins to 
particular antigenic targets, such as tumor cells. Finally, by 
replacement of the immunoglobulin loci of one species with 
that of another, animals of one species have been endowed 
with the capacity to respond to immunization by producing 
antibodies encoded by the donor's genetically transplanted Ig 
genes. By capturing a significant sample of all of the im- 
munoglobulin heavy- and light-chain variable-region genes 
via incorporation into libraries of bacteriophage, it has been 



possible to achieve significant and useful reconstructions of 
the entire antibody repertoires of individuals. The next few 
sections describe each of these types of antibody genetic en- 
gineering. 

Chimeric and Hybrid Monoclonal Antibodies 
Have Potent Clinical Potential 

One approach to engineering an antibody is to clone recom- 
binant DNA containing the promoter, leader, and variable- 
region sequences from a mouse antibody gene and the 
constant-region exons from a human antibody gene (Figure 
5-20). The antibody encoded by such a recombinant gene is a 
mouse-human chimera, commonly known as a humanized 
antibody. Its antigenic specificity, which is determined by the 
variable region, is derived from the mouse DNA; its isotype, 
which is determined by the constant region, is derived from 
the human DNA. Because the constant regions of these 
chimeric antibodies are encoded by human genes, the anti- 



LIGHT-CHAIN GENES 



HEAVY-CHAIN GENES 




n vectors 



lan monoclor 
n heavy- and light-chain exprc 
e produced. These vectors are t 
myeloma cells. Culture in ampicillin medium selects for transfected 
myeloma cells that secrete the chimeric antibody. [Adapted from M. 
Verhoeyen and L Reichmann, 1988, BioEssays 8:74.] 



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bodies have fewer mouse antigenic determinants and are 
far less immunogenic when administered to humans than 
mouse monoclonal antibodies (Figure 5-21a). The ability of 
the mouse variable regions remaining in these humanized 
antibodies to provide the appropriate binding site to allow 
specific recognition of the target antigen has encouraged fur- 
ther exploration of this approach. It is possible to produce 
chimeric human-mouse antibodies in which only the se- 
quences of the CDRs are of mouse origin (Figure 5-2 lb). An- 
other advantage of humanized chimeric antibodies is that 
they retain the biological effector functions of human anti- 
body and are more likely to trigger human complement acti- 
vation or Fc receptor binding. One such chimeric human- 
mouse antibody has been used to treat patients with B-cell 
varieties of non-Hodgkin's lymphoma (see Clinical Focus). 




Heteroconjugate 

and hybrid 

neered by recombinant DNA technology, (a) Chimeric mouse-hu- 
man monoclonal antibody containing the V H and V L domains of a 
mouse monoclonal antibody (blue) and C L and C H domains of a hu- 
man monoclonal antibody (gray), (b) A chimeric monoclonal anti- 
body containing only the CDRs of a mouse monoclonal antibody 
(blue bands) grafted within the framework regions of a human mon- 
oclonal antibody is called a "humanized" monoclonal antibody, (c) A 
chimeric monoclonal antibody in which the terminal Fc domain is re- 
placed by toxin chains (white), (d) A heteroconjugate in which one- 
half of the mouse antibody molecule is specific for a tumor antigen 
and the other half is specific for the CD3/T-cell receptor complex. 



Chimeric monoclonal antibodies that function as im- 
munotoxins (see Figure 4-23) can also be prepared. In this 
case, the terminal constant-region domain in a tumor- 
specific monoclonal antibody is replaced with toxin chains 
(Figure 5-21c). Because these immunotoxins lack the 
terminal Fc domain, they are not able to bind to cells bearing 
Fc receptors. These immunotoxins can bind only to tumor 
cells, making them highly specific as therapeutic reagents. 

Heteroconjugates, or bispecific antibodies, are hybrids 
of two different antibody molecules (Figure 5-21d). They 
can be constructed by chemically crosslinking two different 
antibodies or by synthesizing them in hybridomas consist- 
ing of two different monoclonal-antibody-producing cell 
lines that have been fused. Both of these methods generate 
mixtures of monospecific and bispecific antibodies from 
which the desired bispecific molecule must be purified. Us- 
ing genetic engineering to construct genes that will encode 
molecules only with the two desired specificities is a much 
simpler and more elegant approach. Several bispecific mole- 
cules have been designed in which one half of the antibody 
has specificity for a tumor and the other half has specificity 
for a surface molecule on an immune effector cell, such as an 
NK cell, an activated macrophage, or a cytotoxic T lympho- 
cyte (CTL). Such heteroconjugates have been designed to 
activate the immune effector cell when it is crosslinked to 
the tumor cell so that it begins to mediate destruction of the 
tumor cell. 

Monoclonal Antibodies Can Be Constructed 
from Ig-Gene Libraries 

A quite different approach for generating monoclonal anti- 
bodies employs the polymerase chain reaction (PCR) to am- 
plify the DNA that encodes antibody heavy-chain and 
light-chain Fab fragments from hybridoma cells or plasma 
cells. A promoter region and EcoRl restriction site (see Chap- 
ter 23) are added to the amplified sequences, and the result- 
ing constructs are inserted into bacteriophage \, yielding 
separate heavy- and light-chain libraries. Cleavage with 
EcoRl and random joining of the heavy- and light-chain 
genes yield numerous novel heavy-light constructs (Figure 
5-22). 

This procedure generates an enormous diversity of anti- 
body specificities — libraries with >10 10 unique members 
have been obtained — and clones containing these random 
combinations of H + L chains can be rapidly screened for 
those secreting antibody to a particular antigen. The level of 
diversity is comparable to the human in vivo repertoire, and 
it is possible to demonstrate that specificities against a wide 
variety of antigens can be obtained from these libraries. Such 
a combinatorial library approach opens the possibility of ob- 
taining specific antibodies without any need what 



However, the real challenge to bypassing in vivo immu- 
nization in the derivation of useful antibodies of high affin- 
ity lies in finding ways to mimic the biology of the humoral 



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Generation of B-Cell and T-Cell Respor 



CLINICAL FOCUS 



Therapy for Non-Hodgkin's 
Lymphoma and Other Diseases 
by Genetically Engineered 
Antibodies 



are 
cancers of lymphatic tissue in which the 
tumor cells are of lymphocytic origin. 
There are two major forms of lymphoma: 
Hodgkin's lymphoma and non-Hodgkin's 
lymphoma. The less common form is 
Hodgkin's lymphoma, named for its dis- 
coverer, Thomas Hodgkin, an English 
physician. This unusually gifted early 
pathologist, who worked without the ben- 
efit of a microscope, recognized this con- 
dition in several patients and first 
described the anatomical features of the 
disease in 1832. Because many tissue 
specimens taken from patients Hodgkin 
suspected of harboring the disease were 
saved in the Gordon Museum of Guy's 
Hospital in London, it has been possible 
for later generations to judge the accu- 
racy of his diagnoses. Hodgkin has fared 
well. Studies of these preserved tissues 
confirm that he was right in about 60% of 
the cases, a surprising achievement, con- 
sidering the technology of the time. Actu- 
ally, most lymphoma is non-Hodgkin's 
type and includes about 1 different types 
of disease. B-cell lymphomas are an im- 
portant fraction of these. 

For some years now, the major thera- 
pies directed against lymphomas have 
been radiation, chemotherapy, or a com- 
bination of both. While these therapies 
benefit large numbers of patients by in- 
creasing survival, relapses after treat- 
ment are common, and many treated 
patients experience debilitating side ef- 
fects. The side effects are an expected 
consequence of these therapies, because 
the agents used kill or severely damage a 
broad spectrum of normal cells as well as 
tumor cells. One of the holy grails of can- 
cer treatment is the discovery of therapies 



that will affect only the tumor cells and 
completely spare normal cells. If particu- 
lar types of cancer cells had antigens that 
were tumor specific, these antigens 
would be ideal targets for immune attack. 
Unfortunately, there are few such mole- 
cules known. However, a number of anti- 
gens are known that are restricted to the 
cell lineage in which the tumor originated 
and are expressed on the tumor cells. 

Many cell-lineage-specific antigens 
have been identified for B lymphocytes 
and B lymphomas, including immuno- 
globulin, the hallmark of the B cell, and 
CD20, a membrane-bound phosphopro- 
tein. CD20 has emerged as an attractive 
candidate for antibody-mediated im- 
munotherapy because it is present on B 
lymphomas, and antibody-mediated 
crosslinking does not cause it to down- 
regulate or internalize. Indeed, some 
years ago, mouse monoclonal antibodies 
were raised against CD20, and one of 
these has formed the basis for an anti-B- 
cell lymphoma immunotherapy. This ap- 
proach appears ready to take its place as 
an adjunct or alternative to radiation and 
chemotherapy. The development of this 
anti-tumor antibody is an excellent case 
study of the combined application of im- 

ogy to engineer a novel therapeutic agent. 
The original anti-CD20 antibody was a 
mouse monoclonal antibody with murine 
7 heavy chains and k light chains. The 
DNA sequences of the light- and heavy- 
chain variable regions of this antibody 
were amplified by PCR. Then a chimeric 
gene was created by replacing the CDR 
gene sequences of a human -yl heavy 
chain with those from the murine heavy 
chain. In a similar maneuver, CDRs from 
the mouse k were ligated into a human k 



gene. The chimeric genes thus created 
were incorporated into vectors that per- 
mitted high levels of expression in mam- 
malian cells. When an appropriate cell 
line was co-transfected with both of these 
constructs, it produced chimeric antibod- 
ies containing CDRs of mouse origin to- 
gether with human variable-region 
frameworks and constant regions. After 
purification, the biological activity of the 
antibody was evaluated, first in vitro and 
then in a primate animal model. 

The initial results were quite promis- 
ing. The grafted human constant region 
supported effector functions such as the 
complement-mediated lysis or antibody- 
dependent cell-mediated cytotoxicity 
(ADCC) of human B lymphoid cells. Fur- 
thermore, weekly injections of the anti- 
body into monkeys resulted in the rapid 
and sustained depletion of B cells from pe- 
ripheral blood, lymph nodes, and even 
bone marrow. When the anti-CD20 anti- 
body infusions were stopped, the differen- 
tiation of new B cells from progenitor 
populations allowed B-cell populations 
eventually to recover and approach nor- 
mal levels. From these results, the hope 
grew that this immunologically active 
chimeric antibody could be used to clear 
entire B cell populations, including B lym- 
phoma cells, from the body in a way that 
spared other cell populations. This led to 
the trial of the antibody in human patients. 

The human trials enrolled patients 
with B-cell lymphoma who had a relapse 
after chemotherapy or radiation treat- 
ment. These trials addressed three im- 
portant issues: efficacy, safety, and 
immunogenicity. While not all patients re- 
sponded to treatment with anti-CD20, 
close to 50% exhibited full or partial re- 
mission. Thus, efficacy was demon- 
strated, because this level of response is 
comparable to the success rate with tradi- 
tional approaches that employ highly cyto- 
toxic drugs or radiation — it offers a truly 
alternative therapy. Side effects such as 
nausea, low blood pressure, and short- 
ness of breath were seen in some pa- 
tients (usually during or shortly after the 
initiation of therapy); these were, for the 
most part, not serious or life-threatening. 
Consequently, treatment with the 



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and Expression of Immunoglobulin Genes chaf 



chimeric anti-CD20 appears safe. Patients 
who received the antibody have been ob- 
served closely for the appearance of hu- 
man anti-mouse-lg antibodies (HAMA) 
and for human anti-chimeric antibody 
(HACA) responses. Such responses were 
not observed. Therefore, the antibody was 
not immunogenic. The absence of such 
responses demonstrate that antibodies 
can be genetically engineered to mini- 
mize, or even avoid, untoward immune 
reactions. Another reason for humanizing 
mouse antibodies arises from the very 
short half life (a few hours) of mouse IgG 
antibodies in humans compared with the 
three-week half lives oftheir human or hu- 
manized counterparts. 

Antibody engineering has also con- 
tributed to the therapy of other malignan- 
cies such as breast cancer, which is 
diagnosed in more than 180,000 Ameri- 
can women each year. A little more than a 
quarter of all breast cancer patients have 



cancers that over-express a growth factor 
receptor called HER2 (human epidermal 
growth factor receptor 2). Many tumors 
that over-express HER2 grow faster and 
pose a more serious threat than those with 
normal levels of this protein on their sur- 
face. A chimeric anti-HER2 monoclonal 
antibody in which all of the protein except 
the CDRs are of human origin was created 
by genetic engineering. Specifically, the 
DNA sequences for the heavy-chain and 
light-chain CDRs were taken from cloned 
mouse genes encoding an anti-HER2 
monoclonal antibody. As in the anti-CD20 
strategy described above, each of the 
mouse CDR gene segments were used to 
replace the corresponding human CDR 
gene segments in human genes encoding 
the human IgG-i heavy chain and the hu- 
man k light chain. When this engineered 
antibody is used in combination with a 
chemotherapeutic drug, it is highly effec- 
tive against metastatic breast cancer. The 



effects on patients who were given only a 
chemotherapeutic drug were compared 
with those for patients receiving both the 
chemotherapeutic drug and the engi- 
neered anti-HER2 antibody. The combina- 
tion anti-HER2/chemotherapy treatment 
showed significantly reduced rates of tu- 
mor progression, a higher percentage of 
responding patients, and a higher one-year 
survival rate. Treatment with Herceptin, as 
this engineered monoclonal antibody is 
called, has become part of the standard 
repertoire of breast cancer therapies. 

The development of engineered and 
conventional monoclonal antibodies is 
one of the most active areas in the phar- 
maceutical industry. The table provides a 
partial compilation of monoclonal anti- 
bodies that have received approval from 
the Food and Drug Administration 
(FDA) for use in the treatment of human 
disease. Many more are in various 
stages of development and testing. 



Monoclonal 
antibody [mAB] 
(Product Name) 


Nature of 
antibody 




Target 

(antibody specificity) 


Treatment for 


Muromonab-CD3 
(Orthoclone OKT3) 


Mouse mAB 




T cells 

(CD3, a T cell antigen) 


Acute rejection of liver, heart 
and kidney transplants 


Abciximab 
(ReoPro) 


chimeric 




Clotting receptor of platelets 
(GPIIb/llla) 


Blood clotting during angioplasty 
and other cardiac procedures 


Daclizumab 
(Zenapax) 


Humanized 


nAB 


Activated T cells 

(IL-2 receptor alpha subunit) 


Acute rejection of 
kidney transplants 


Infliximab 
(Remicade) 


chimeric 




Tumor necrosis factor, (TNF) a 
mediator of inflammation. (TNF) 


Rheumatoid arthritis 
and Crohn's disease 


Palivizumab 
(Synagis) 


Humanized 


nAB 


Respiratory Syncytial Virus (RSV) 
(F protein, a component of RSV) 


RSV infection in 

children, particularly infants 


Gemtuzumab 
(Mylotarg) 


Humanized 


nAB 


Many cells of the myeloid lineage 
(CD33, an adhesion molecule) 


Acute myeloid 
leukemia (AML) 


Alemtuzumab 
(Campath) 


Humanized 


nAB 


Many types of leukocytes 
(CD52 a cell surface antigen) 


B cell chronic 
lymphocytic leukemia 


Trastuzumab 
(Herceptin) 


Humanized 


nAB 


An epidermal growth factor 
receptor (HER2 receptor) 


HER2 receptor-positive 
advanced breast cancers 


Rituximab 
(Rituxan) 


Humanized 


nAB 


B cells 

(CD20 a B cell surface antigen) 


Relapsed or refractory 
non-Hodgkins lymphoma 


(Zevalin) 


Mouse mAB 




(CD20, a B cell surface antigen) 


Relapsed or refractory 
non-Hodgkins lymphoma 


SOURCE: Adapted from P. Ca 


Iter. 2001. Improving 


the efficac 


of antibody-based cancer therapies.™. 


«4*/Gw!«r1:118. 



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Generation of B-Cell and T-Cell Respor 





"T^ 



light- and heavy- ™ ' 



Prepare random 
combinational 



-f-H 

I V H -C H 1 

Promoter 



at I EcoRl 



Not I EcoKl 



leral procedure for producing gene libraries e 
coding Fab fragments. In this procedure, isolated mRNA that e 
codes heavy and light chains is amplified by the polymerase cha 
reaction (PCR) and cloned in X vectors. Random combinations 
heavy- and light-chain genes generate an enormous number 
heavy-light constructs encoding Fab fragments with different an 
genie specificity. [Adapted from W. D. Huse et al., 1989, Scien 
246:1275.] 



immune response. As we shall see in Chapter 11, the in vivo 
evolution of most humoral immune responses produces two 
desirable outcomes. One is class switching, in which a variety 
of antibody classes of the same specificity are produced. This 
is an important consideration because the class switching 
that occurs during an immune response produces antibodies 
that have the same specificity but different effector functions 
and hence, greater biological versatility. The other is the gen- 
eration of antibodies of higher and higher affinity as the re- 
sponse progresses. A central goal of Ig-gene library ap- 
proaches is the development of strategies to produce anti- 
bodies of appropriate affinity in vitro as readily as they are 
generated by an in vivo immune response. When the formi- 
dable technical obstacles to the achievement of these goals 
are overcome, combinatorial approaches based on phage 



libraries will allow the routine and widespread production 
of useful antibodies from any desired species without the 
limitations of immunization and hybridoma technology 
that currently complicate the production of monoclonal 
antibodies. 

Mice Have Been Engineered with 
Human Immunoglobulin Loci 

It is possible to functionally knock out, or disable, the heavy- 
and light-chain immunoglobulin loci in mouse embryonic 
stem (ES) cells. N. Lonberg and his colleagues followed this 
procedure and then introduced large DNA sequences (as 
much as 80 kb) containing human heavy- and light- chain 
gene segments. The DNA sequences contained constant-re- 
gion gene segments, J segments, many V-region segments, 
and, in the case of the heavy chain, D H segments. The ES cells 
containing these miniature human Ig gene loci (miniloci) are 
used to derive lines of transgenic mice that respond to anti- 
genic challenge by producing antigen-specific human anti- 
bodies (Figure 5-23). Because the human heavy- and 
light-chain miniloci undergo rearrangement and all the 
other diversity-generating processes, such as N-addition, P- 
addition, and even somatic hypermutation after antigenic 
challenge, there is an opportunity for the generation of a 
great deal of diversity in these mice. The presence of human 
heavy-chain minilocus genes for more than one isotype and 
their accompanying switch sites allows class switching as 
well. A strength of this method is that these completely hu- 
man antibodies are made in cells of the mouse B-cell lineage, 
from which antibody-secreting hybridomas are readily de- 
rived by cell fusion. This approach thus offers a solution to 
the problem of producing human monoclonal antibodies of 
any specificity desired. 



■ Immunoglobulin k and X light chains and heavy chai 
encoded by three separate multigene families, each 
taining numerous gene segments and located on dif 



■ Functional light-chain and heavy-chain genes are gener- 
ated by random rearrangement of the variable-region gene 
segments in germ-line DNA. 

■ V(D)J joining is catalyzed by the recombinase activiating 
genes, RAG-1 and RAG-2, and the participation of other 
enzymes and proteins. The joining of segments is directed 
by recombination signal sequences (RSS), conserved DNA 
sequences that flank each V, D, and J gene segment. 

■ Each recombination signal sequence contains a conserved 
heptamer sequence, a conserved nonamer sequence, and 
either a 12-bp (one-turn) or 23-bp (two-turn) spacer. 
During rearrangement, gene segments flanked by a one- 
turn spacer join only to segments flanked by a two-turn 
spacer, assuring proper V L -J L and V h -D h -Jh joining. 



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V H genes D genes J genes C^ Cyl 

CXXX^HHHHHHMHHHHM^O 

Germ-line human heavy-chain minilocus 



Jk 8 enes C K 

xxx: WHHH^ — 



Human miniloci 



M*M [HHHH D 



YYYY 



into mice. The capacity of mice to rearrange Ig heavy- and light- 
chain gene segments was disabled by knocking out the C^ and C K 
loci. The antibody-producing capacity of these mice was reconsti- 
tuted by introducing long stretches of DNA incorporating a large 
part of the human germ-line k and heavy-chain loci (miniloci). 



iring both heavy- 



antibody specific for the target 
Nature 368:856.] 



establish a line of transgenic mice 
light-chain human miniloci. 
ults in the production of human 
antigen. [N. Lonberg et al., 1994, 



i Immunoglobulin gene rearrangements occur in sequential 
order, heavy-chain rearrangements first, followed by light- 
chain rearrangements. Allelic exclusion is a consequence of 
the functional rearrangement of the immunoglobulin 
DNA of only one parental chromosome and is necessary to 
assure that a mature B cell expresses immunoglobulin with 
a single antigenic specificity. 

i The major sources of antibody diversity, which can gener- 
ate >10 10 possible antibody combining sites, are: random 



joining of multiple V, J, and D germ-line gene segments; 
random association of heavy and light chains; junctional 
flexibility; P-addition; N-addition; and somatic mutation. 

i After antigenic stimulation of mature B cells, class switch- 
ing results in expression of different classes of antibody 
(IgG, IgA, and IgE) with the same antigenic specificity. 

i Differential RNA processing of the immunoglobulin 
heavy-chain primary transcript generates membrane- 
bound antibody in mature B cells, secreted antibody in 



Go to www.whfreeman.ee 
Review and quiz of key ter 



© = 



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Generation of B-Cell and T-Cell Respor 



q of IgM a 



plasma cells, and the simultaneous expre 
IgD by mature B cells. 

■ Transcription of immunoglobulin genes is regulated by 
three types of DNA regulatory sequences: promoters, en- 
hancers, and silencers. 

■ Growing knowledge of the molecular biology of im- 
munoglobulin genes has made it possible to engineer anti- 
bodies for research and therapy. The approaches include 
chimeric antibodies, bacteriophage-based combinatorial 
libraries of Ig-genes, and the transplantation of extensive 
segments of human Ig loci into mice. 

References 

Chen, J., Y. Shinkai, F. Young, and F. W. Alt. 1994. Probing im- 
mune functions in RAG-deficient mice. Curr. Opin. Immunol. 
6:313. 

Cook, G. P., and I. M. Tomlinson. 1995. The human im- 
munoglobulin V H repertoire. Immunol. Today 16:237. 

Dreyer, W. J., and J. C. Bennett. 1965. The molecular basis of an- 
tibody formation. Proc. Natl. Acad. Sci. U.S.A. 54:864. 

Fugmann, S. D., I. L. Lee, P. E. Shockett, I. J. Villey, and D. G. 
Schatz. 2000. The RAG proteins and V(D)J recombination: 
Complexes, ends and transposition. Annu. Rev. Immunol. 
18:495. 

Gavilondo, J. V., and J. W. Larrick. 2000. Antibody engineering at 
the millennium. Biotechniques 29:128. 

Hayden, M. S., L. K. Gilliand, and J. A. Ledbetter. 1997. Antibody 
engineering. Curr. Opin. Immunol. 9:201. 

Hesslein, D. G, and D. G Schatz. 2001. Factors and forces con- 
trolling V(D)J recombination. Adv. Immunol. 78:169. 

Hozumi, N., and S. Tonegawa. 1976. Evidence for somatic re- 
arrangement of immunoglobulin genes coding for variable 
and constant regions. Proc. Natl. Acad. Sci. U.S.A. 73:3628. 

Maloney, D. G, et al. 1997. IDEC-C2B8 (Rituximab) anti-CD20 
monoclonal antibody therapy in patients with relapsed low- 
grade non-Hodgkin's lymphoma. Blood 90:2188. 

Manis, J. P., M. Tian, and F. W. Alt. 2002. Mechanism and control 
of class-switch recombination. Trends Immunol. 23:31. 

Matsuda, E, K. Ishii, P. Bourvagnet, Ki Kuma, H. Hayashida, T 
Miyata, and T. Honjo. 1998. The complete nucleotide sequence 
of the human immunoglobulin heavy chain variable region lo- 
cus. /. Exp. Med. 188:2151. 

Max, E. E. 1998. Immunoglobulins: molecular genetics. In Fun- 
damental Immunology, 4th ed., W. E. Paul, ed. Lippincott- 
Raven, Philadelphia. 

Mills, F. C, N. Harindranath, M. Mitchell, and E. E. Max. 1997. 
Enhancer complexes located downstream of both human im- 
munoglobulin C alpha genes. /. Exp. Med. 186:845. 

Oettinger, M. A., et al. 1990. RAG-1 and RAG-2, adjacent genes 
that synergistically activate V(D)J recombination. Science 
248:1517. 



of antibody diversity. 



Tonegawa, S. 1983. Somatic generatio 
Nature 302:575. 

Van Gent, D. C, et al. 1995. Initiation of V(D)J recombin 
a cell-free system. Cell 81:925. 



Winter, G., : 
349:293. 



id C. Milstein. 1990. Man-made antibodies. Nature 



c.uk/imt-doc/public/ 



USEFUL WEB SITES 

http://www.rn rc-cpe.< 
INTRO.html#maps 

V BASE: This database and informational site is maintained at 
the MRC Centre for Protein Engineering in England. It is an 
excellent and comprehensive directory of information on the 
human germ-line variable region. 

http://www.mgen.uni-heidelberg.de/SD/SDscFvSite.html 

The Recombinant Antibody Page: This site has a number of 
links that provide interesting opportunities to explore the po- 
tential of genetic engineering of antibodies. 



.ebi.ac.uk/imgt/hla/intro.htm 



http://w 

The IMGT site contains a collection of databases of genes rel- 
evant to the immune system. The IMGT/LIGM database 
houses sequences belonging to the immunoglobulin super- 
family and of T cell antigen receptor sequences. 



Study Questions 



Clinical Focus Question The Clinical Focus section includes a 
table of monoclonal antibodies approved for clinical use. Two, 
Rituxan and Zevalin, are used for the treatment of non- 
Hodgkins lymphoma. Both target CD20, a B-cell surface antigen. 
Zevalin is chemically modified by attachment of radioactive iso- 
topes (yttrium-90, a (3 emitter or indium-Ill, a high energy y 
emitter) that lethally irradiate cells to which the monoclonal an- 
tibody binds. Early experiments found that Zevalin without a ra- 
dioactive isotope attached was an ineffective therapeutic agent, 
whereas unlabeled Rituxan, a humanized mAB, was effective. 
Furthermore, Rituxan with a radioactive isotope attached was 
too toxic; Zevalin bearing the same isotope in equivalent 
amounts was far less toxic. Explain these results. (Hint: The 
longer a radioactive isotope stays in the body, the greater the dose 
of radiation absorbed by the body.) 

1 . Indicate whether each of the following statements is true or 
false. If you think a statement is false, explain why. 

a. V K gene segments sometimes join to Qv gene segments. 

b. With the exception of a switch to IgD, immunoglobulin 
class switching is mediated by DNA rearrangements. 

c. Separate exons encode the transmembrane portion of 
each membrane immunoglobulin. 

d. Although each B cell carries two alleles encoding the im- 
munoglobulin heavy and light chains, only one allele is 
expressed. 



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' Immunology 5e: 



. Primary transcripts a 
by removal of intron: 



e processed into functional mRNA 
capping, and addition of a poly-A 



:arrangement of variable- 
ight-chain gene segments in 



f. The primary transcript is an RNA complem 
coding strand of the DNA and includes both ii 



2. Explain why a V H segment cannot join directly with a T H seg- 
ment in heavy-chain gene rearrangement. 

3. Considering only combinatorial joining of gene segments 
and association of light and heavy chains, how many differ- 
ent antibody molecules potentially could be generated from 
germ-line DNA containing 500 V L and 4 J L gene segments 
and 300 V H , 15 D H , and 4 T H gene segments? 



below (a-g), select the 
More than 



. For each incomplet 
phrase(s) that correctly complet 
one choice may be correct. 



iglobulin gene segments 



( 1 ) promote Ig diversification 

(2) assemble a complete Ig coding sequence 

(3) allow changes in coding information dui 



(4) increase the affinity of immunoglobulin for antibody 

(5) all of the above 



of immunoglobulin genes 
for 

(1) allelic exclusion 

(2) class switching from IgM to IgG 

(3) affinity maturation 

(4) all of the above 

(5) none of the above 



c. The frequency of somatic n 
during 

( 1 ) differentiation of pre-B cells into mature B cells 

(2) differentiation of pre-T cells into mature T cells 

(3) generation of memory B cells 

(4) antibody secretion by plasma cells 

(5) none of the above 

d. Kappa and lambda light-chain genes 

( 1 ) are located on the same chromosome 

(2) associate with only one type of heavy chain 

(3) can be expressed by the same B cell 

(4) all of the above 

(5) none of the above 

e. Generation of combinatorial diversity among im- 
munoglobulins involves 

(1) mRNA splicing 

(2) DNA rearrangement 

(3) recombination signal sequences 

(4) one-turn/two-turn joining rule 

(5) switch sites 

f. A B cell becomes immunocompetent 

(1) following productive rearrangement of variable- 
region heavy-chain gene segments in germ-line DNA 






(2) following prodi 
region heavy- ch; 
germ-line DNA 

(3) following class 

(4) during affinity 

(5) following binding of T H cytokines to their receptors 
on the B cell 

g. The mechanism that permits immunoglobulins to be 
synthesized in either a membrane-bound or secreted 

( 1 ) allelic exclusion 

(2) codominant expression 

(3) class switching 

(4) the one-turn/two-turn joining rule 

(5) differential RNA processing 

5. What mechanisms generate the three hypervariable regions 
(complementarity-determining regions) of immunoglobu- 
lin heavy and light chains? Why is the third hypervariable re- 
gion (CDR3) more variable than the other two (CDR1 and 
CDR2)? 

6. You have been given a cloned myeloma cell line that secretes 
IgG with the molecular formula 72^2- Both the heavy and 
light chains in this cell line are encoded by genes derived 
from allele 1. Indicate the form(s) in which each of the genes 
listed below would occur in this cell line using the following 
symbols: G = germ line form; R = productively rearranged 
form; NP = nonproductively rearranged form. State the rea- 
son for your choice in each case. 

a. Heavy-chain allele 1 d. K-chain allele 2 

b. Heavy-chain allele 2 e. \-chain allele 1 

c. K-chain allele 1 f. \-chain allele 2 

7. You have a B-cell lymphoma that has made nonproductive 
rearrangements for both heavy-chain alleles. What is the 
arrangement of its light-chain DNA? Why? 

8. Indicate whether each of the class switches indicated below 
can occur (Yes) or cannot occur (No). 



1 IgD 


d. IgAtoIgG 


>IgA 


e. IgM to IgG 


IgG 





9. Describe one advantage and one disadvantage of N- 
nucleotide addition during the rearrangement of im- 
munoglobulin heavy-chain gene segments. 

1 0. X-ray crystallographic analyses of many antibody molecules 
bound to their respective antigens have revealed that the 
CDR3 of both the heavy and light chains make contact with 
the epitope. Moreover, sequence analyses reveal that the 
variability of CDR3 is greater than that of either CDR1 or 
CDR2. What mechanisms account for the greater diversity 
in CDR3? 

1 1 . How many chances does a developing B cell have to generate 
a functional immunoglobulin light-chain gene? 

1 2. Match the terms below (a-h) to the description(s) that fol- 
low (1-11). Each description may be used once, more than 
once, or not at all; more than one description may apply to 



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Terms 






(8) 


a. RAG-landRAG-2 


e. 


P-nucleotides 




b. Double-strand break 


f. 


N-nucleotides 


(9) 


repair (DSBR) enzymes 


g- — 


Promoters 


(101 


c. Coding joints 


h. __ 


Enhancers 




d. RSSs 









Descriptions 

(1) Junctions between immunoglobulin gene segments 
formed during rearrangement 

(2) Source of diversity in antibody heavy chains 

(3) DNA regulatory sequences 

(4) Conserved DNA sequences, located adjacent to V, D, 
and J segments, that help direct gene rearrangement 

(5) Enzymes expressed in developing B cells 

(6) Enzymes expressed in mature B cells 

(7) Nucleotide sequences located close to each leader seg- 
ment in immunoglobulin genes to which RNA poly- 



Product of endonuclease cleavage of hairpin interme- 
diates in Ig-gene rearrangement 
Enzymes that are defective in SCID mice 
Nucleotide sequences that greatly increase the rate of 
transcription of rearranged immunoglobulin genes 
compared with germ-line DNA 
(11) Nucleotides added by TdT enzyme 

13. Many B-cell lymphomas express surface immunoglobulin 
on their plasma membranes. It is possible to isolate this lym- 
phoma antibody and make a high affinity, highly specific 
mouse monoclonal anti-idiotype antibody against it. What 
steps should be taken to make this mouse monoclonal anti- 
body most suitable for use in the patient. Is it highly likely 
that, once made, such an engineered antibody will be gener- 
ally useful for lymphoma patients? 



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chapter 6 



Antigen-Antibody 
Interactions: 

Principles and Applications 



THE ANTIGEN-ANTIBODY INTERACTION IS A BIMO- 
lecular association similar to an enzyme-substrate 
interaction, with an important distinction: it does 
not lead to an irreversible chemical alteration in either the 
antibody or the antigen. The association between an anti- 
body and an antigen involves various noncovalent interac- 
tions between the antigenic determinant, or epitope, of the 
antigen and the variable-region ( V H /V L ) domain of the an- 
tibody molecule, particularly the hypervariable regions, or 
complementarity-determining regions (CDRs). The exquis- 
ite specificity of antigen-antibody interactions has led to the 
development of a variety of immunologic assays, which can 
be used to detect the presence of either antibody or antigen. 
Immunoassays have played vital roles in diagnosing diseases, 
monitoring the level of the humoral immune response, and 
identifying molecules of biological or medical interest. 
These assays differ in their speed and sensitivity; some are 
strictly qualitative, others are quantitative. This chapter ex- 
amines the nature of the antigen-antibody interaction, and it 
describes various immunologic assays that measure or ex- 
ploit this interaction. 



Strength of Antigen-Antibody 
Interactions 

The noncovalent interactions that form the basis of antigen- 
antibody (Ag-Ab) binding include hydrogen bonds, ionic 
bonds, hydrophobic interactions, and van der Waals interac- 
tions (Figure 6-1). Because these interactions are individu- 
ally weak (compared with a covalent bond), a large number 
of such interactions are required to form a strong Ag-Ab in- 
teraction. Furthermore, each of these noncovalent interac- 
tions operates over a very short distance, generally about 
1 X 10~ 7 mm (1 angstrom, A); consequently, a strong Ag- 
Ab interaction depends on a very close fit between the anti- 
gen and antibody. Such fits require a high degree of 
complementarity between antigen and antibody, a require- 
ment that underlies the exquisite specificity that character- 
izes antigen-antibody interactions. 




■ Strength of Antigen-Antibody Interactions 

■ Cross-Reactivity 

■ Precipitation Reactions 

■ Agglutination Reactions 

■ Radioimmunoassay 

■ Enzyme-Linked Immunosorbent Assay 

■ Western Blotting 

■ Immunoprecipitation 

■ Immunofluorescence 

■ Flow Cytometry and Fluorescence 

■ Alternatives to Antigen-Antibody Reactions 

■ Immunoelectron Microscopy 



Antibody Affinity Is a Quantitative Measure 
of Binding Strength 

The combined strength of the noncovalent interactions be- 
tween a single antigen-binding site on an antibody and a sin- 
gle epitope is the affinity of the antibody for that epitope. 
Low-affinity antibodies bind antigen weakly and tend to dis- 
sociate readily, whereas high-affinity antibodies bind antigen 
more tightly and remain bound longer. The association be- 
tween a binding site on an antibody (Ab) with a monovalent 
antigen (Ag) can be described by the equation 

h 



Ag + Ab ^ 



± Ag-Ab 



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VISUALIZING CONCEPTS 

ANTIGEN 



-CH 2 -OH ••• 0=C — CH 2 —CH 2 — Hydrogen bond 

-CH 2 -CH 2 -NH 3 + O x 

C -CH 2 -CH 2 - Ionic bond 



- Hydrophobic 
interactions 




CH=— CH— CH 2 — 



CH— CH 3 CHj-CH— 



[Jgj/Si, 



- CH 2 — C +H 3 N— CH 2 — Ionic bond 



If The interaction between an antibody and an anti- 
gen depends on four types of noncovalent forces: (1) hydrogen 
bonds, in which a hydrogen atom is shared between two elec- 
tronegative atoms; (2) ionic bonds between oppositely charged 
residues; (3) hydrophobic interactions, in which water forces hy- 



drophobic groups together; and (4) van der Waals 
between the outer electron clouds of two or more i 



queous environment, nor 


covalent interactions are e 


xtremely 


veak and depend upon do 


e complementarity of the s 


napes of 


ntibody and antigen. 







where fcj is the forward (association) rate constant and k_ x 
the reverse (dissociation) rate constant. The ratio k 1 /k_ 1 
the association constant K a (i.e., k 1 /k_ 1 = K a ), a measure < 
affinity. Because K a is the equilibrium constant for the abo^ 
reaction, it can be calculated from the ratio of the molar coi 
centration of bound Ag-Ab complex to the molar ci 
tions of unbound antigen and antibody at equilibri 
follows: 



K a 



[Ag-Ab] 



[Ab][Ag] 

The value of K a varies for different Ag-Ab complexes and 
depends upon both fc 1; which is expressed in units of 
liters/mole/second (L/mol/s), and fc_ l5 which is expressed in 
units of 1 /second. For small haptens, the forward rate con- 
stant can be extremely high; in some cases, k x can be as high 
as 4 X 10 8 L/mol/s, approaching the theoretical upper limit 
of diffusion-limited reactions (10 9 L/mol/s). For larger pro- 
tein antigens, however, k x is smaller, with values in the range 
of 10 5 L/mol/s. 

The rate at which bound antigen leaves an antibody's 
binding site (i.e., the dissociation rate constant, k- X ) plays a 
major role in determining the antibody's affinity for an 
antigen. Table 6-1 illustrates the role of k^ 1 in determining 



the association constant K a for several Ag-Ab interactions. 
For example, the k x for the DNP-L-lysine system is about 
one fifth that for the fluorescein system, but its k- i is 200 
times greater; consequently, the affinity of the antifluores- 
cein antibody K a for the fluorescein system is about 1000- 
fold higher than that of anti-DNP antibody. Low-affinity 
Ag-Ab complexes have K a values between 10 4 and 10 5 
L/mol; high-affinity complexes can have K a values as high 
aslO n L/mol. 

For some purposes, the dissociation of the antigen-anti- 
body complex is of interest: 

Ag-Ab ; " Ab + Ag 

The equilibrium constant for that reaction is K d , the recipro- 
cal of K a 

K d = [Ab][Ag]/[Ab-Ag] = \/K a 



ind is a quantitative indicate] 
:omplex; very stable complext 
md less stable ones have highe 
The affinity constant, K a , c; 
•ium dialysis or by various ne\ 



i dialysis 



of the stability of an Ag-Ab 
> have very low values of K d , 
values. 

n be determined by equilib- 
r er methods. Because equilib- 



for many the standard against which 



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Anti-DNP 

Anti-flue 

Anti-bovine serum albumin (BSA) 

SOURCE: Adapted from H. N. Eisen, 1990, 



Ligand k, 

€-DNP-L-lysine 8 X 10 7 

Fluorescein 4 X 10 8 

Dansyl-BSA 3 X 10 5 



other methods are evaluated, it is described here. This proce- 
dure uses a dialysis chamber containing two equal compart- 
ments separated by a semipermeable membrane. Antibody is 
placed in one compartment, and a radioactively labeled lig- 
and that is small enough to pass through the semipermeable 
membrane is placed in the other compartment (Figure 6-2). 
Suitable ligands include haptens, oligosaccharides, and oligo- 
peptides. In the absence of antibody, ligand added to com- 
partment B will equilibrate on both sides of the membrane 
(Figure 6-2a). In the presence of antibody, however, part 



of the labeled ligand will be bound to the antibody at equi- 
librium, trapping the ligand on the antibody side of the ves- 
sel, whereas unbound ligand will be equally distributed in 
both compartments. Thus the total concentration of ligand 
will be greater in the compartment containing antibody (Fig- 
ure 6-2b). The difference in the ligand concentration in the 
two compartments represents the concentration of ligand 
bound to the antibody (i.e., the concentration of Ag-Ab com- 
plex). The higher the affinity of the antibody, the more ligand 
is bound. 



Control: No antibody present 

(ligand equilibrates on both sides equally) 



A B A B 

• . * ~* • 

•:•• • • . • 




Experimental: Antibody in A 

ibrium more ligand in A due to Ab binding) 



i 


B 
• •• 

• • 

• •• 


— > 


A 

■V: 


B 

• 



Radiolabeled 



ysis. (a) The dialysis chamber contains two compartments (A and 
separated by a semipermeable membrane. Antibody is added to oi 
compartment and a radiolabeled ligand to another. At equilibria 
ion of radioactivity in both compartments is me 




Time, h 

sured. (b) Plot of concentration of ligand in each compartment with 
time. At equilibrium, the difference in the concentration of radioac- 
tive ligand in the two compartments represents the amount of ligand 
bound to antibody. 



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Since the total concentration of antibody in the equilib- 
lm dialysis chamber is known, the equilibrium equation 
n be rewritten as: 



K a = [Ab-Ag]/[Ab][Ag] = 



c(n - r) 
ofboi 



where r equals the ratio of the 

to total antibody concentration. 

ligand, and n is the number of binding sites j 

molecule. This expression can be rearranged 

Scatchard equation: 



tibody 



-= K a n 



' A',;- 



Values for r and c can be obtained by repeating the equi- 
librium dialysis with the same concentration of antibody but 
with different concentrations of ligand. If K.^ is a constant, 
that is, if all the antibodies within the dialysis chamber have 
the same affinity for the ligand, then a Scatchard plot of r/c 
versus r will yield a straight line with a slope of — K a (Figure 
6-3a). As the concentration of unbound ligand c increases, r/c 
approaches 0, and r approaches n, the valency, equal to the 
number of binding sites per antibody molecule. 

Most antibody preparations are polyclonal, and K. d is 
therefore not a constant because a heterogeneous mixture of 
antibodies with a range of affinities is present. A Scatchard 
plot of heterogeneous antibody yields a curved line whose 



slope is constantly changing, reflecting this antibody hetero- 
geneity (Figure 6-3b). With this type of Scatchard plot, it is 
possible to determine the average affinity constant, K , by de- 
termining the value of K a when half of the antigen-binding 
sites are filled. This is conveniently done by determining the 
slope of the curve at the point where half of the antigen bind- 
ing sites are filled. 

Antibody Avidity Incorporates Affinity 
of Multiple Binding Sites 

The affinity at one binding site does not always reflect the 
true strength of the antibody-antigen interaction. When 
complex antigens containing multiple, repeating antigenic 
determinants are mixed with antibodies containing multiple 
binding sites, the interaction of an antibody molecule with 
an antigen molecule at one site will increase the probability 
of reaction between those two molecules at a second site. The 
strength of such multiple interactions between a multivalent 
antibody and antigen is called the avidity. The avidity of an 
antibody is a better measure of its binding capacity within bi- 
ological systems (e.g., the reaction of an antibody with anti- 
genic determinants on a virus or bacterial cell) than the 
affinity of its individual binding sites. High avidity can com- 
pensate for low affinity. For example, secreted pentameric 



(a) Homogeneous antibody 



Ob) Heterogeneous antibody 





| Scatchard pit 
dialyses with a c< 
centration of ligand. In these 
and/mole antibody and c is the 
Scatchard plot, both the equilibi 
binding sites per antibody mo 
ained. (a) If 



based on repeated equilibrium 
of antibody and varying con- 
equals moles of bound lig- 
of free ligand. From a 
constant (Q and the number of 
e (n), or its valency, can be ob- 
: same affinity, then a Scatchard 



plot yields a straight line with a slope of-/< a . Thex intercept is n, the 
valency of the antibody, which is 2 for IgC and other divalent Igs. For 
IgM, which is pentameric, n = 10, and for dimeric IgA, n = 4. In this 



graph, antibody #1 has a higher affinity than antibody #2. (b) If the 
antibody preparation is polyclonal and has a range of affinities, a 
Scatchard plot yields a curved line whose slope is constantly chang- 
ing. The average affinity constant K can be calculated by determin- 
ing the value of /< a when half ofthe binding sites are occupied (i.e., 
when r = 1 in this example). In this graph, antiserum #3 has a higher 
affinity (/< = 2.4 X 10 8 ) than antiserum #4 (K = 1.25 X 10 8 ). Note 
that the curves shown in (a) and (b) are for divalent antibodies such 
as IgC. 



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IgM often has a lower affinity than IgG, but the high avidity 
of IgM, resulting from its higher valence, enables it to bind 
antigen effectively. 



Cross-Reactivity 

Although Ag-Ab reactions are highly specific, in some cases 
antibody elicited by one antigen can cross-react with an un- 
related antigen. Such cross-reactivity occurs if two different 
antigens share an identical or very similar epitope. In the lat- 
ter case, the antibody's affinity for the cross-reacting epitope 
is usually less than that for the original epitope. 

Cross-reactivity is often observed among polysaccharide 
antigens that contain similar oligosaccharide residues. The 
ABO blood-group antigens, for example, are glycoproteins 
expressed on red blood cells. Subtle differences in the termi- 
nal residues of the sugars attached to these surface proteins 
distinguish the A and B blood-group antigens. An individual 
lacking one or both of these antigens will have serum anti- 
bodies to the missing antigen(s). The antibodies are induced 
not by exposure to red blood cell antigens but by exposure to 
cross-reacting microbial antigens present on common in- 
testinal bacteria. These microbial antigens induce the for- 
mation of antibodies in individuals lacking the similar 
blood-group antigens on their red blood cells. (In individu- 
als possessing these antigens, complementary antibodies 
would be eliminated during the developmental stage in 
which antibodies that recognize self epitopes are weeded 
out.) The blood-group antibodies, although elicited by mi- 
crobial antigens, will cross-react with similar oligosaccha- 
rides on foreign red blood cells, providing the basis for 
blood typing tests and accounting for the necessity of com- 
patible blood types during blood transfusions. A type A in- 
dividual has anti-B antibodies; a type B individual has 
anti-A; and a type O individual thus has anti-A and anti-B 
(Table 6-2). 

A number of viruses and bacteria have antigenic determi- 
nants identical or similar to normal host-cell components. In 
some cases, these microbial antigens have been shown to 
elicit antibody that cross-reacts with the host-cell compo- 
nents, resulting in a tissue-damaging autoimmune reaction. 



Antigens on RBCs 



AandB 
Neither 



The bacterium Streptococcus pyogenes, for example, expresses 
cell-wall proteins called M antigens. Antibodies produced to 
streptococcal M antigens have been shown to cross-react 
with several myocardial and skeletal muscle proteins and 
have been implicated in heart and kidney damage following 
streptococcal infections. The role of other cross-reacting 
antigens in the development of autoimmune diseases is dis- 
cussed in Chapter 20. 

also exhibit cross-reactivity. For instance, 



epitopes with variola "\ 
This cross-reactivity w 



;es cowpox, expi 
is, the 



tioned in Chapter 1. 



:sses cross-reacting 
agent of smallpox, 
er's method of us- 
smallpox, as men- 



Precipitation Reactions 

Antibody and soluble antigen interacting in aqueous solu- 
tion form a lattice that eventually develops into a visible pre- 
cipitate. Antibodies that aggregate soluble antigens are called 
precipitins. Although formation of the soluble Ag-Ab com- 
plex occurs within minutes, formation of the visible precipi- 
tate occurs more slowly and often takes a day or two to reach 

Formation of an Ag-Ab lattice depends on the valency of 
both the antibody and antigen: 

■ The antibody must be bivalent; a precipitate will not 
form with monovalent Fab fragments. 

■ The antigen must be either bivalent or polyvalent; that is, 
it must have at least two copies of the same epitope, or 
have different epitopes that react with different 
antibodies present in polyclonal antisera. 

Experiments with myoglobin illustrate the requirement 
that protein antigens be bivalent or polyvalent for a precip- 
itin reaction to occur. Myoglobin precipitates well with spe- 
cific polyclonal antisera but fails to precipitate with a specific 
monoclonal antibody because it contains multiple, distinct 
epitopes but only a single copy of each epitope (Figure 6-4a). 
Myoglobin thus can form a crosslinked lattice structure with 
polyclonal antisera but not with monoclonal antisera. The 
principles that underlie precipitation reactions are presented 
because they are essential for an understanding of commonly 
used immunological assays. Although various modifications 
of the precipitation reaction were at one time the major types 
of assay used in immunology, they have been largely replaced 
by methods that are faster and, because they are far more sen- 
sitive, require only very small quantities of antigen or anti- 
body. Also, these modern assay methods are not limited to 
antigen-antibody reactions that produce a precipitate. Table 
6-3 presents a comparison of the sensitivity, or minimum 
amount of antibody detectable, by a number of immunoas- 
says. 



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POLYCLONAL ANTISERUM 








Precipitali 
form lattices, or large a^ 



However, if ea 

ognized by a given monocle 
two molecules of antigen an 
itation curve for a system of i 
of the amount of 
concentrations (at 



(a) Polyclonal antibodies can 
5, that precipitate out of solution. 
: contains only a single epitope rec- 
ntibody, the antibody can link only 
precipitate is formed, (b) A precip- 
ntigen and its antibodies. This plot 
body precipitated versus increasing antigen 
body) reveals three zones: a 



Antigen added 



zone of antibody 
body not bound 



n which precipitation is inhibited and anti- 
;n can be detected in the supernatant; an 
nee zone of maximal precipitation in which antibody and 
ntigen form large insoluble complexes and neither antibody nor 
ntigen can be detected in the supernatant; and a zone of antigen ex- 
ess in which precipitation is inhibited and antigen not bound to 
ntibody can be detected in the supernatant. 



Precipitation Reactions in Fluids Yield 
a Precipitin Curve 

A quantitative precipitation reaction can be performed by 
placing a constant amount of antibody in a series of tubes 
and adding increasing amounts of antigen to the tubes. At 
one time this method was used to measure the amount of 
antigen or antibody present in a sample of interest. After the 
precipitate forms, each tube is centrifuged to pellet the pre- 
cipitate, the supernatant is poured off, and the amount of 
precipitate is measured. Plotting the amount of precipitate 
against increasing antigen concentrations yields a precipitin 
curve. As Figure 6-4b shows, excess of either antibody or 
antigen interferes with maximal precipitation, which occurs 
in the so-called equivalence zone, within which the ratio of 
antibody to antigen is optimal. As a large multimolecular 
lattice is formed at equivalence, the complex increases in size 
and precipitates out of solution. As shown in Figure 6-4, un- 
der conditions of antibody excess or antigen excess, extensive 
lattices do not form and precipitation is inhibited. Although 
the quantitative precipitation reaction is seldom used exper- 



imentally today, the principles of antigen excess, antibody ex- 
cess, and equivalence apply to many Ag-Ab reactions. 

Precipitation Reactions in Gels Yield 
Visible Precipitin Lines 

Immune precipitates can form not only in solution but also in 
an agar matrix. When antigen and antibody diffuse toward one 
another in agar, or when antibody is incorporated into the agar 
and antigen diffuses into the antibody-containing matrix, a 
visible line of precipitation will form. As in a precipitation re- 
action in fluid, visible precipitation occurs in the region of 
equivalence, whereas no visible precipitate forms in regions of 
antibody or antigen excess. Two types of immunodiffusion re- 
actions can be used to determine relative concentrations of an- 
tibodies or antigens, to compare antigens, or to determine the 
relative purity of an antigen preparation. They are radial im- 
munodiffusion (the Mancini method) and double immun- 
odiffusion (the Ouchterlony method); both are carried out in 
a semisolid medium such as agar. In radial immunodiffusion, 
an antigen sample is placed in a well and allowed to diffuse into 



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RADIAL IMMUNODIFFUSION 



Sensitivity* 
(n-gantibody/m 



Precipitation reaction in 


fluids 


20-200 


Precipitation reactions i 


i gels 




Mancini radial immunodiffusion 


10-50 


Ouchterlony double i 


nmunodiffusion 


20-200 


Immunoelectrophore 


sis 


20-200 


Rocket electrophores 




2 


Agglutination reactions 






Direct 




0.3 


Passive agglutination 




0.006-0.06 


Agglutination inhibit! 




0.006-0.06 


Radioimmunoassay 




0.0006-0.006 


Enzyme-linked immunosorbent 




assay (ELISA) 




<0.0001-0.01 


ELISA using chemilumi 




<0.0001-0.01" r 


Immunofluorescence 




1.0 


Flow cytometry 




0.06-0.006 



SOURCE: Adapted from 




DOUBLE IMMUNODIFFUSION 




* Diagrammatic representation of radial immunodiffu- 
n (Mancini method) and double immunodiffusion (Ouchterlony 



gen (red) diffuse 



method) in a gel. In both c 


ases, large insoluble c 


omplex 


the agar in the zone of equ 


valence, visible as lin 


esofp 


(purple regions). Only the 


ntigen (red) diffuses 


n radia 


diffusion, whereas both the 


antibody (blue) and ar 


tigen ( 


in double immunodiffusion 







agar containing a suitable dilution of an antiserum. As the 
antigen diffuses into the agar, the region of equivalence is es- 
tablished and a ring of precipitation, a precipitin ring, forms 
around the well (Figure 6-5, upper panel). The area of the pre- 
cipitin ring is proportional to the concentration of antigen. By 
comparing the area of the precipitin ring with a standard curve 
(obtained by measuring the precipitin areas of known concen- 
trations of the antigen), the concentration of the antigen sam- 
ple can be determined. In the Ouchterlony method, both 
antigen and antibody diffuse radially from wells toward each 
other, thereby establishing a concentration gradient. As equiv- 
alence is reached, a visible line of precipitation, a precipitin 
line, forms (Figure 6-5, lower panel). 

Immunoelectrophoresis Combines 
Electrophoresis and Double 
Immunodiffusion 

In Immunoelectrophoresis, the antigen mixture is first elec- 
trophoresed to separate its components by charge. Troughs 
are then cut into the agar gel parallel to the direction of 



the electric field, and antiserum is added to the troughs. 
Antibody and antigen then diffuse toward each other and 
produce lines of precipitation where they meet in appropri- 
ate proportions (Figure 6-6a). Immunoelectrophoresis is 
used in clinical laboratories to detect the presence or absence 
of proteins in the serum. A sample of serum is elec- 
trophoresed, and the individual serum components are 
identified with antisera specific for a given protein or im- 
munoglobulin class (Figure 6-6b). This technique is useful in 
determining whether a patient produces abnormally low 
amounts of one or more isotypes, characteristic of certain 
immunodeficiency diseases. It can also show whether a pa- 
tient overproduces some serum protein, such as albumin, 
immunoglobulin, or transferrin. The immunoelectropho- 
retic pattern of serum from patients with multiple myeloma, 
for example, shows a heavy distorted arc caused by the large 
amount of myeloma protein, which is monoclonal Ig and 
therefore uniformly charged (Figure 6-6b). Because Immu- 
noelectrophoresis is a strictly qualitative technique that only 
detects relatively high antibody concentrations (greater than 
several hundred |jLg/ml), it utility is limited to the detection 



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144 part ii Generation of B-Cell and T-Cell Respons 









Qjg Immunoelectrophoresis of an antigen mixture. 
(a) An antigen preparation (orange) is first electrophoresed, which 
separates the component antigens on the basis of charge. Antiserum 
(blue) is then added to troughs on one or both sides of the separated 
antigens and allowed to diffuse; in time, lines of precipitation (col- 
ored arcs) form where specific antibody and antigen interact, (b) Im- 
munoelectrophoretic patterns of human serum from a patient with 
myeloma. The patient produces a large amount of a monoclonal IgG 



IgA 
IgG 
IgM 

K 

X 


= 



(X-light-chain-bearing) antibody. A sample of serum from the patient 
was placed in the well of the slide and electrophoresed. Then anti- 
serum specific for the indicated antibody class or light chain type was 
placed in the top trough of each slide. At the concentrations of pa- 
tient's serum used, only anti-lgC and anti-X antibodies produced 
lines of precipitation. [Part(b), Robert A. Kyle and Terry A. Katzman, 
Manual of Clinical Immunology, 1997, N. Rose, ed., ASM Press, Wash- 
ington, D.C., p. 164.] 



of quantitative abnormalities only when the departure from 
normal is striking, as in immunodeficiency states and im- 
munoproliferative disorders. 

A related quantitative technique, rocket electrophore- 
sis, does permit measurement of antigen levels. In rocket 
electrophoresis, a negatively charged antigen is elec- 
trophoresed in a gel containing antibody. The precipitate 
formed between antigen and antibody has the shape of a 
rocket, the height of which is proportional to the concen- 
tration of antigen in the well. One limitation of rocket 
electrophoresis is the need for the antigen to be negatively 
charged for electrophoretic movement within the agar 
matrix. Some proteins, immunoglobulins for example, 



are not sufficiently charged to be quantitatively analyzed 
by rocket electrophoresis; nor is it possible to measure 
the amounts of several antigens in a mixture at the same 



Agglutination Reactions 

The interaction between antibody and a particulate antigen re- 
sults in visible clumping called agglutination. Antibodies that 
produce such reactions are called agglutinins. Agglutination 
reactions are similar in principle to precipitation reactions; 
they depend on the crosslinking of polyvalent antigens. Just as 



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an excess of antibody inhibits precipitation reactions, such 
excess can also inhibit agglutination reactions; this inhibition 
is called the prozone effect. Because prozone effects can be en- 
countered in many types of immunoassays, understanding the 
basis of this phenomenon is of general importance. 

Several mechanisms can cause the prozone effect. First, at 
high antibody concentrations, the number of antibody bind- 
ing sites may greatly exceed the number of epitopes. As a re- 
sult, most antibodies bind antigen only univalently instead 
of multivalently. Antibodies that bind univalently cannot 
crosslink one antigen to another. Prozone effects are readily 
diagnosed by performing the assay at a variety of antibody 
(or antigen) concentrations. As one dilutes to an optimum 
antibody concentration, one sees higher levels of agglutina- 
tion or whatever parameter is measured in the assay being 
used. When one is using polyclonal antibodies, the prozone 
effect can also occur for another reason. The antiserum may 
mtibodies that bind to the 
induce agglutination; these antibodies, 
itibodies, are often of the IgG class. At 
of IgG, incomplete antibodies may oc- 
igenic sites, thus blocking access by IgM, 
iod agglutinin. This effect is not seen with agglu- 
noclonal antibodies. The lack of agglutinating 
i incomplete antibody may be due to restricted 
the hinge region, making it difficult for the anti- 
the required angle for optimal cross-linking 
two or more particulate antigens. Alterna- 
tively, the density of epitope distribution or the location of 
some epitopes in deep pockets of a particulate antigen may 
make it difficult for the antibodies specific for these epitopes 
to agglutinate certain particulate antigens. When feasible, the 
solution to both of these problems is to try different antibod- 
ies that may react with other epitopes of the antigen that do 
not present these limitations. 

Hemagglutination Is Used in Blood Typing 

Agglutination reactions (Figure 6-7) are routinely performed 
to type red blood cells (RBCs). In typing for the ABO 



high 

called incomplete 
high concentratioi 
cupy most of the ai 
which is a good ags 



activity of 
flexibility i: 

of epitopes 




| Demonstration of hemagglutination using antibodies 
against sheep red blood cells (SRBCs). The control tube (10) con- 
tains only SRBCs, which settle into a solid "button." The experimen- 
tal tubes 1-9 contain a constant number of SRBCs plus serial 
two-fold dilutions of anti-SRBC serum. The spread pattern in the ex- 
perimental series indicates positive hemagglutination through tube 
3. [Louisiana State University Medical Center /MIP. Courtesy of Harriet 
C. W. Thompson.] 



antigens, RBCs are mixed on a slide with antisera to the A 
or B blood-group antigens. If the antigen is present on the 
cells, they agglutinate, forming a visible clump on the slide. 
Determination of which antigens are present on donor and 
recipient RBCs is the basis for matching blood types for 
transfusions. 



Bacterial Agglutination Is Used 
To Diagnose Infection 

A bacterial infection often elicits the production of serum 
antibodies specific for surface antigens on the bacterial cells. 
The presence of such antibodies can be detected by bacterial 
agglutination reactions. Serum from a patient thought to be 
infected with a given bacterium is serially diluted in an array 
of tubes to which the bacteria is added. The last tube showing 
visible agglutination will reflect the serum antibody titer ol 
the patient. The agglutinin titer is defined as the reciprocal ol 
the greatest serum dilution that elicits a positive agglutina 
tion reaction. For example, if serial twofold dilutions ol 
serum are prepared and if the dilution of 1/640 shows agglu 
tination but the dilution of 1/1280 does not, then the agglu 
tination titer of the patient's serum is 640. In some cases 
serum can be diluted up to 1/50,000 and still show agglutina- 
tion of bacteria. 

The agglutinin titer of an antiserum can be used to diag- 
nose a bacterial infection. Patients with typhoid fever, for ex- 
ample, show a significant rise in the agglutination titer to 
Salmonella typhi. Agglutination reactions also provide a way 
to type bacteria. For instance, different species of the bac- 
terium Salmonella can be distinguished by agglutination re- 
actions with a panel of typing antisera. 

Passive Agglutination Is Useful 
with Soluble Antigens 

The sensitivity and simplicity of agglutination reactions can 
be extended to soluble antigens by the technique of passive 
hemagglutination. In this technique, antigen-coated red 
blood cells are prepared by mixing a soluble antigen with red 
blood cells that have been treated with tannic acid or 
chromium chloride, both of which promote adsorption of 
the antigen to the surface of the cells. Serum containing anti- 
body is serially diluted into microtiter plate wells, and the 
antigen-coated red blood cells are then added to each well; 
agglutination is assessed by the size of the characteristic 
spread pattern of agglutinated red blood cells on the bottom 
of the well, like the pattern seen in agglutination reactions 
(see Figure 6-7). 

Over the past several years, there has been a shift away 
from red blood cells to synthetic particles, such as latex 
beads, as matrices for agglutination reactions. Once the anti- 
gen has been coupled to the latex beads, the preparation can 
either be used immediately or stored for later use. The use 
of synthetic beads offers the advantages of consistency, 



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' Immunology 5e: 



ofB-CellandT-Cell Respons 



uniformity, and stability. Furthermore, a 
tions employing synthetic beads can be read rapidly, often 
within 3 to 5 minutes of mixing the beads with the test sam- 
ple. Whether based on red blood cells or the more convenient 
and versatile synthetic beads, agglutination reactions are 
simple to perform, do not require expensive equipment, and 
can detect small amounts of antibody (concentrations as low 
as nanograms per mi 

In Agglutination Inhibition, Absence of 
Agglutination Is Diagnostic of Antigen 

A modification of the agglutination reaction, called agglu- 
tination inhibition, provides a highly sensitive assay for 
small quantities of an antigen. For example, one of the early 



types of home pregnancy test kits included latex particles 
coated with human chorionic gonadotropin (HCG) and 
antibody to HCG (Figure 6-8). The addition of urine from 
a pregnant woman, which contained HCG, inhibited agglu- 
tination of the latex particles when the anti-HCG antibody 
was added; thus the absence of agglutination indicated 
pregnancy. 

Agglutination inhibition assays can also be used to deter- 
mine whether an individual is using certain types of illegal 
drugs, such as cocaine or heroin. A urine or blood sample is 
first incubated with antibody specific for the suspected drug. 
Then red blood cells (or other particles) coated with the drug 
are added. If the red blood cells are not agglutinated by the 
antibody, it indicates the sample contained an antigen recog- 
nized by the antibody, suggesting that the individual was 



KIT REAGENTS 

hcg< VyC and 



Hapten carrier-conjugate 



TEST PROCEDURE 



Incubate H CG c; 



Observe for visible 



POSSIBLE REACTIONS 

(p) reaction: not pregnant 



© 
• •• 



• •' 






•& - •& 



-> A G Visible clumping 



V 



ff* The original home pregnancy test kit employed hap- 
n inhibition to determine the presence or absence of human chori- 
nic gonadotropin (HCG). The original test kits used the presence or 
jsence of visible clumping to determine whether HCG was pre 



s not pregnant, her urine 



>uld n 



HCG; i 



kit would react, producing visible clumping. If a woman was preg- 
nant, the HCG in her urine would bind to the anti-HCG antibodies, 
thus inhibiting the subsequent binding of the antibody to the HCG- 
carrier conjugate. Because of this inhibition, no visible clumping oc- 






s pregn; 



t. The kits currently on the market u 



;, the anti-HCG antibodies and HCG-carrier conjugate in the EUSA-based assays (see Figure 6-10). 



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8536d_ch06_137-160 8/1/02 9:01 AM 



' Immunology 5e: 



;: Principles and Application; 



using the illicit drug. One problem with these tests is that 
some legal drugs have chemical structures similar to those of 
illicit drugs, and these legal drugs may cross-react with the 
antibody, giving a false-positive reaction. For this reason a 
positive reaction must be confirmed by a nonimmunologic 
method. 

Agglutination inhibition assays are widely used in clinical 
laboratories to determine whether an individual has been 
exposed to certain types of viruses that cause agglutination of 
red blood cells. If an individual's serum contains specific an- 
tiviral antibodies, then the antibodies will bind to the virus 
and interfere with hemagglutination by the virus. This tech- 
nique is commonly used in premarital testing to determine 
i with respect to rubella virus. 
im dilution to show inhibition 
is the titer of the serum. A titer 
) indicates that 
er of less than 10 is 



e status of 
The reciprocal of the last 
of rubella hemaggli 
greater than 10(l:10dilu 
mune to rubella, whereas 



a lack of immunity a 
rubella v 



id the need for 



with the 



Radioimmunoassay 



One of the most sensitive techniques for detecting antigen or 
antibody is radioimmunoassay (RIA). The technique was 
first developed in 1960 by two endocrinologists, S. A. Berson 
and Rosalyn Yalow, to determine levels of insulin-anti-in- 
sulin complexes in diabetics. Although their technique en- 
countered some skepticism, it soon proved its value for 
measuring hormones, serum proteins, drugs, and vitamins at 
concentrations of 0.001 micrograms per milliliter or less. In 
1977, some years after Berson's death, the significance of the 
technique was acknowledged by the award of a Nobel Prize to 

The principle of RIA involves competitive binding of ra- 
diolabeled antigen and unlabeled antigen to a high-affinity 
antibody. The labeled antigen is mixed with antibody at a 
concentration that saturates the antigen-binding sites of the 
antibody. Then test samples of unlabeled antigen of un- 
known concentration are added in progressively larger 
amounts. The antibody does not distinguish labeled from 
unlabeled antigen, so the two kinds of antigen compete for 
available binding sites on the antibody. As the concentration 
of unlabeled antigen increases, more labeled antigen will be 
displaced from the binding sites. The decrease in the amount 



of radiolabeled 
presence of the 1 
the amount of a 
The antigen 
isotope such as 
t( 3 H) 



bound to specific antibody in the 

t sample is measured in order to determine 

igen present in the test sample. 

generally labeled with a gamma-emitting 

1/5 I, but beta-emitting isotopes such as tri- 

also routinely used as labels. The radiola- 



beled antigen is part of the assay mixture; the test sample 
may be a complex mixture, such as serum or other body 
fluids, that contains the unlabeled antigen. The first step in 



setting up an RIA is to determine the amount of antibody 
needed to bind 50%-70% of a fixed quantity of radioactive 
antigen (Ag A ) in the assay mixture. This ratio of antibody 
to Ag* is chosen to ensure that the number of epitopes 
presented by the labeled antigen always exceeds the total 
number of antibody binding sites. Consequently, unlabeled 
antigen added to the sample mixture will compete with ra- 
diolabeled antigen for the limited supply of antibody. Even 
a small amount of unlabeled antigen added to the assay 
mixture of labeled antigen and antibody will cause a de- 
crease in the amount of radioactive antigen bound, and this 
decrease will be proportional to the amount of unlabeled 
antigen added. To determine the amount of labeled antigen 
bound, the Ag-Ab complex is precipitated to separate it 
from free antigen (antigen not bound to Ab), and the ra- 
dioactivity in the precipitate is measured. A standard curve 
can be generated using unlabeled antigen samples of 
known concentration (in place of the test sample), and 
from this plot the amount of antigen in the test mixture 
may be precisely determined. 

Several methods have been developed for separating the 
bound antigen from the free antigen in RIA. One method in- 
volves precipitating the Ag-Ab complex with a secondary 
anti-isotype antiserum. For example, if the Ag-Ab complex 
contains rabbit IgG antibody, then goat anti-rabbit IgG will 
bind to the rabbit IgG and precipitate the complex. Another 
method makes use of the fact that protein A of Staphylococcus 
aureus has high affinity for IgG. If the Ag-Ab complex con- 
tains an IgG antibody, the complex can be precipitated by 
mixing with formalin-killed S. aureus. After removal of the 
complex by either of these methods, the amount of free la- 
beled antigen remaining in the supernatant can be measured 
in a radiation counter; subtracting this value from the total 
amount of labeled antigen added yields the amount of la- 
beled antigen bound. 

Various solid-phase RIAs have been developed that make 
it easier to separate the Ag-Ab complex from the unbound 
antigen. In some cases, the antibody is covalently cross- 
linked to Sepharose beads. The amount of radiolabeled anti- 
gen bound to the beads can be measured after the beads have 
been centrifuged and washed. Alternatively, the antibody can 
be immobilized on polystyrene or polyvinylchloride wells 
and the amount of free labeled antigen in the supernatant 
can be determined in a radiation counter. In another ap- 
proach, the antibody is immobilized on the walls of mi- 
croliter wells and the amount of bound antigen determined. 
Because the procedure requires only small amounts of sam- 
ple and can be conducted in small 96-well microtiter plates 
(slightly larger than a 3 X 5 card), this procedure is well 
suited for determining the concentration of a particular anti- 
gen in large numbers of samples. For example, a microtiter 
RIA has been widely used to screen for the presence of the he- 
patitis B virus (Figure 6-9). RIA screening of donor blood has 
sharply reduced the incidence of hepatitis B infections in re- 
cipients of blood transfusions. 



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8536d_ch06_137-160 8/1/02 9:01 AM 



' Immunology 5e: 



ofB-CellandT-Cell Respons 



Infected serum [ 12 5l] HBsAg [ 12 5l] HBsAg Uninfected 

?\ /•'; 'S 7° 



Unlabeled 
HBsAg 



\vv4\ Kv-v^l 



olid-phase radioimmunoassay (RIA) to detect 
>patitis B virus in blood samples, (a) Microtiter wells are coated 
th a constant amount of antibody specific for HBsAg, the surface 
itigen on hepatitis B virions. A serum sample and [ 125 l]HBsAg 
e then added. After incubation, the supernatant is removed and 
e radioactivity of the antigen-antibody complexes is measured. If 
e sample is infected, the amount of label bound will be less than 




Concentration of unlabeled HBsAg, ng/ml 

in controls with uninfected serum, (b) A standard curve is obtained 
by adding increasing concentrations of unlabeled HBsAg to a fixed 
quantity of [ 125 l]HBsAg and specific antibody. From the plot of the 
percentage of labeled antigen bound versus the concentration of 
unlabeled antigen, the concentration of HBsAg in unknown serum 
samples can be determined by using the linear part of the curve. 



Enzyme- Linked Immunosorbent 
Assay 

Enzyme-linked immunosorbent assay, commonly known 
as ELISA (or EIA), is similar in principle to RIA but depends 
on an enzyme rather than a radioactive label. An enzyme 
conjugated with an antibody reacts with a colorless substrate 
to generate a colored reaction product. Such a substrate is 
called a chromogenic substrate. A number of enzymes have 
been employed for ELISA, including alkaline phosphatase, 
horseradish peroxidase, and (3-galactosidase. These assays 
approach the sensitivity of RIAs and have the advantage of 
being safer and less costly. 

There Are Numerous Variants of ELISA 

A number of variations of ELISA have been developed, al- 
lowing qualitative detection or quantitative measurement 
of either antigen or antibody. Each type of ELISA can be 
used qualitatively to detect the presence of antibody or 
antigen. Alternatively, a standard curve based on known 



concentrations of antibody or antigen is prepared, from 
which the unknown concentration of a sample can be 
determined. 

INDIRECT ELISA 

Antibody can be detected or quantitatively determined with 
an indirect ELISA (Figure 6-10a). Serum or some other sam- 
ple containing primary antibody (AbJ is added to an anti- 
gen-coated microtiter well and allowed to react with the 
antigen attached to the well. After any free Abi is washed 
away, the presence of antibody bound to the antigen is de- 
tected by adding an enzyme-conjugated secondary anti-iso- 
type antibody (Ab 2 ), which binds to the primary antibody. 
Any free Ab 2 then is washed away, and a substrate for the en- 
zyme is added. The amount of colored reaction product that 
forms is measured by specialized spectrophotometric plate 
readers, which can measure the absorbance of all of the wells 
of a 96-well plate in seconds. 

Indirect ELISA is the method of choice to detect the pres- 
ence of serum antibodies against human immunodeficiency 
virus (HIV), the causative agent of AIDS. In this assay, re- 
combinant envelope and core proteins of HIV are adsorbed 



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8536d_ch06_137-160 8/1/02 9:01 AM 



' Immunology 5e: 



(a) Indirect ELISA 



;: Principles and Applications chapter 6 149 






-1^ 




^ 




A h 


i\ 




Add specific 

antibody to be 

measured 




Add enzyme- 
conjugated 
secondary 




Add substrate (S 



lY Yl 



bod 




Add enzyme- 
secondary antibody 








ations in the enzyme-linked immunosorbent as- 
say (ELISA) technique allow determination of antibody or antigen. 
Each assay can be used qualitatively, or quantitatively by comparison 
with standard curves prepared with known concentrations of anti- 
body or antigen. Antibody can be determined with an indirect ELISA 





Add enzyme- 




Add substrate 






and measure 


secondary 




color 


antibody 






(a), whereas antigen can be det 


ermined 


vith a sandwich ELISA (b) or 


competitive ELISA (c). In the c 


ompetitiv 


e ELISA, which is an inhibi- 


tion-type assay, the concentrat' 


onofanti 


gen is inversely proportional 


to the color produced. 







as solid-phase antigens to microtiter wells. Individuals in- 
fected with HIV will produce serum antibodies to epitopes on 
these viral proteins. Generally, serum antibodies to HIV can 
be detected by indirect ELISA within 6 weeks of infection. 

SANDWICH ELISA 

Antigen can be detected or measured by a sandwich ELISA 
(Figure 6-10b). In this technique, the antibody (rather than 
the antigen) is immobilized on a microtiter well. A sample 
containing antigen is added and allowed to react with the 
immobilized antibody. After the well is washed, a second en- 
zyme-linked antibody specific for a different epitope on the 
antigen is added and allowed to react with the bound anti- 
gen. After any free second antibody is removed by washing, 



substrate is added, and the colored reaction product is 
measured. 

COMPETITIVE ELISA 

Another variation for measuring amounts of antigen is com- 
petitive ELISA (Figure 6-10c). In this technique, antibody is 
first incubated in solution with a sample containing antigen. 
The antigen-antibody mixture is then added to an antigen- 
coated microtiter well. The more antigen present in the sam- 
ple, the less free antibody will be available to bind to the 
antigen-coated well. Addition of an enzyme-conjugated sec- 
ondary antibody (Ab 2 ) specific for the isotype of the primary 
antibody can be used to determine the amount of primary 
antibody bound to the well as in an indirect ELISA. In the 



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8536d_ch06_137-160 8/1/02 12:41PM Page 150 



ofB-CeilandT-Cell Respons 



competitive assay, however, the higher the concentration of 
antigen in the original sample, the lower the absorbance. 



CHEMILUMINESCENCE 

Measurement of light produced by chemiluminescence dur- 
ing certain chemical reactions provides a convenient and 
highly sensitive alternative to absorbance measurements in 
ELISA assays. In versions of the ELISA using chemilumines- 
cence, a luxogenic (light-generating) substrate takes the place 
of the chromogenic substrate in conventional ELISA reac- 
tions. For example, oxidation of the compound luminol by 
H 2 2 and the enzyme horseradish peroxidase (HRP) pro- 
duces light: 

luminol + H 2 2 
Ab-HRP + Ag >Ab-HRP-Ag > light 

The advantage of chemiluminescence assays over chro- 
mogenic ones is enhanced sensitivity. In general, the detec- 
tion limit can be increased at least ten-fold by switching from 
a chromogenic to a luxogenic substrate, and with the addi- 
tion of enhancing agents, more than 200-fold. In fact, under 
ideal conditions, as little as 5 X 10~ 18 moles (5 attomoles) of 
target antigen have been detected. 

ELISPOT ASSAY 

A modification of the ELISA assay called the ELISPOT assay 
allows the quantitative determination of the number of cells 
in a population that are producing antibodies specific for a 
given antigen or an antigen for which one has a specific anti- 
body (Figure 6-11). In this approach, the plates are coated 
with the antigen (capture antigen) recognized by the anti- 
body of interest or with the antibody (capture antibody) spe- 
cific for the antigen whose production is being assayed. A 
suspension of the cell population under investigation is then 
added to the coated plates and incubated. The cells settle 
onto the surface of the plate, and secreted molecules reactive 
with the capture molecules are bound by the capture mole- 
cules in the vicinity of the secreting cells, producing a ring of 
antigen-antibody complexes around each cell that is produc- 
ing the molecule of interest. The plate is then washed and an 
enzyme-linked antibody specific for the secreted antigen or 
specific for the species (e.g., goat anti-rabbit) of the secreted 
antibody is added and allowed to bind. Subsequent develop- 
ment of the assay by addition of a suitable chromogenic or 
chemiluminescence-producing substrate reveals the position 
of each antibody- or antigen-producing cell as a point of 
color or light. 



Well coated with 




Western Blotting 



Identification of a specific protein in a complex mixture of 
proteins can be accomplished by a technique known as West- 
ern blotting, named for its similarity to Southern blotting, 



In the ELISPOT assay, a well is coated with antibody 
against the antigen of interest, a cytokine in this example, and then a 
suspension of a cell population thought to contain some members syn- 
thesizing and secreting the cytokine are layered onto the bottom of the 
well and incubated. Most of the cytokine molecules secreted by a par- 
ticular cell react with nearby well-bound antibodies. After the incubation 
period, the well is washed and an enzyme-labeled anti-cytokine antibody 
is added. After washing away unbound antibody, a chromogenic sub- 
strate that forms an insoluble colored product is added. The colored 
product (purple) precipitates and forms a spot only on the areas of the 
well where cytokine-secreting cells had been deposited. By counting 
the number of colored spots, it is possible to determine how many 
cytokine-secreting cells were present in the added cell suspension. 



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8536d_ch06_137-160 8/1/02 12:41PM Page 151 



;: Principles and Application; 



(a) Add SDS-treated protein 
to well of gel 







n Western blotting, a protein mixture is (a) treated 
with SDS, a strong denaturing detergent, (b) then separated by elec- 
trophoresis in an SDS polyacrylamide gel (SDS-PAGE) which sepa- 
rates the components according to their molecular weight; lower 
molecular weight components migrate farther than higher molecular 
weight ones, (c) The gel is removed from the apparatus and applied 
to a protein-binding sheet of nitrocellulose or nylon and the proteins 
in the gel are transferred to the sheet by the passage of an electric 
current, (d) Addition of enzyme-linked antibodies detects the antigen 
of interest, and (e) the position of the antibodies is visualized by 
means of an ELISA reaction that generates a highly colored insoluble 
product that is deposited at the site of the reaction. Alternatively, a 
chemiluminescent ELISA can be used to generate light that is readily 
detected by exposure of the blot to a piece of photographic film. 



which detects DNA fragments, and Northern blotting, which 
detects mRNAs. In Western blotting, a protein mixture is 
electrophoretically separated on an SDS-polyacrylamide gel 
(SDS-PAGE), a slab gel infused with sodium dodecyl sulfate 
(SDS), a dissociating agent (Figure 6-12). The protein bands 
are transferred to a nylon membrane by electrophoresis and 
the individual protein bands are identified by flooding the 
nitrocellulose membrane with radiolabeled or enzyme- 
linked polyclonal or monoclonal antibody specific for the 
protein of interest. The Ag-Ab complexes that form on the 
band containing the protein recognized by the antibody can 
be visualized in a variety of ways. If the protein of interest was 
bound by a radioactive antibody, its position on the blot can 
be determined by exposing the membrane to a sheet of x-ray 
film, a procedure called autoradiography. However, the most 
generally used detection procedures employ enzyme-linked 
antibodies against the protein. After binding of the enzyme- 
antibody conjugate, addition of a chromogenic substrate that 
produces a highly colored and insoluble product causes the 
appearance of a colored band at the site of the target antigen. 
The site of the protein of interest can be determined with 
much higher sensitivity if a chemiluminescent compound 
along with suitable enhancing agents is used to produce light 
at the antigen site. 

Western blotting can also identify a specific antibody in a 
mixture. In this case, known antigens of well-defined molec- 
ular weight are separated by SDS-PAGE and blotted onto ni- 
trocellulose. The separated bands of known antigens are then 
probed with the sample suspected of containing antibody 
specific for one or more of these antigens. Reaction of an an- 
tibody with a band is detected by using either radiolabeled or 
enzyme-linked secondary antibody that is specific for the 
species of the antibodies in the test sample. The most widely 
used application of this procedure is in confirmatory testing 
for HIV, where Western blotting is used to determine 
whether the patient has antibodies that react with one or 
more viral proteins. 



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8536d_ch06_137-160 8/1/02 12:41 PM Page 152 n 



ofB-CeilandT-Cell Respons 



Immunoprecipitation 

The immunoprecipitation technique has the advantage of al- 
lowing the isolation of the antigen of interest for further 
analysis. It also provides a sensitive assay for the presence of a 
particular antigen in a given cell or tissue type. An extract pro- 
duced by disruption of cells or tissues is mixed with an anti- 
body against the antigen of interest in order to form an 
antigen-antibody complex that will precipitate. However, if 
the antigen concentration is low (often the case in cell and tis- 
sue extracts), the assembly of antigen-antibody complexes 
into precipitates can take hours, even days, and it is difficult to 
isolate the small amount of immunoprecipitate that forms. 

Fortunately, there are a number of ways to avoid these 
limitations. One is to attach the antibody to a solid support, 
such as a synthetic bead, which allows the antigen-antibody 
complex to be collected by centrifugation. Another is to add 
a secondary antibody specific for the primary antibody to 
bind the antigen-antibody complexes. If the secondary anti- 
body is attached to a bead, the immune complexes can be 
collected by centrifugation. A particularly ingenious version 
of this procedure involves the coupling of the secondary an- 
tibody to magnetic beads. After the secondary antibody 
binds to the primary antibody, immunoprecipitates are 
collected by placing a magnet against the side of the tube 
(Figure 6-13). 

When used in conjunction with biosynthetic radioisotope 
labeling, immunoprecipitation can also be used to determine 



whether a particular antigen is actually synthesized by a cell 
or tissue. Radiolabeling of proteins synthesized by cells of in- 
terest can be done by growing the cells in cell-culture 
medium containing one or more radiolabeled amino acids. 
Generally, the amino acids used for this application are those 
most resistant to metabolic modification, such as leucine, 
cysteine, or methionine. After growth in the radioactive 
medium, the cells are lysed and subjected to a primary anti- 
body specific for the antigen of interest. The Ag-Ab complex 
is collected by immunoprecipitation, washed free of unin- 
corporated radiolabeled amino acid and other impurities, 
and then analyzed. The complex can be counted in a scintil- 
lation counter to obtain a quantitative determination of the 
amount of the protein synthesized. Further analysis often in- 
volves disruption of the complex, usually by use of SDS and 
heat, so that the identity of the immunoprecipitated antigen 
can be confirmed by checking that its molecular weight is 
that expected for the antigen of interest. This is done by sep- 
aration of the disrupted complex by SDS-PAGE and subse- 
quent autoradiography to determine the position of the 
radiolabeled antigen on the gel. 



Immunofluorescence 

In 1944, Albert Coons showed that antibodies could be la- 
beled with molecules that have the property of fluorescence. 
Fluorescent molecules absorb light of one wavelength 



ft 



>^ 



Specific o 



Antigen if 



4 



i 





Add secondary 


Apply magnet 


extract 


antibody 
coupled 


remo ve ° 










beads 






lunoprecipitates can be collected using mag- 
netic beads coupled to a secondary antibody, (a) Treatment of a cell 
extract containing antigen A (red) with a mouse anti-A antibody 
(blue) results in the formation of antigen-antibody complexes, 
(b) Addition of magnetic beads to which a rabbit anti-mouse anti- 
body is linked binds the antigen-antibody complexes (and any unre- 
acted mouse Ig). (c) Placing a magnet against the side of the tube 



allows the rapid collection of the antigen-antibody complexes. After 

plexes can be dissociated and the antigen studied, (d) An electron 
micrograph showing a cell with magnetic beads attached to its sur- 
face via antibodies. [Part (d), P. Croscurth, Institute of Anatomy, Uni- 
versity ofZurich-lrchel.] 



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8536d_ch06_137-160 8/1/02 9:01 AM Page 153 mac79 Mac 7 9 : 45 JW : p^dsby et al . / Immunology 5e: 



;: Principles and Application; 



(excitation) and emit light of another wavelength (emission). fluorescence (546 n 
If antibody molecules are tagged with a fluorescent dye, or a longer wavelength than fluorescein, it can be used in 
fluorochrome, immune complexes containing these fluores- two-color immunofluorescence assays. An antibody 
cently labeled antibodies (FA) can be detected by colored specific to one determinant is labeled with fluorescein, 
light emission when excited by light of the appropriate wave- and an antibody recognizing a different antigen is 
length. Antibody molecules bound to antigens in cells or tis- labeled with rhodamine. The location of the 
sue sections can similarly be visualized. The emitted light can fluorescein-tagged antibody will be visible by its yellow- 
be viewed with a fluorescence microscope, which is equipped green color, easy to distinguish from the red color 
with a UV light source. In this technique, known as im- emitted where the rhodamine-tagged antibody has 
munofluorescence, fluorescent compounds such as fluores- bound. By conjugating fluorescein to one antibody and 
cein and rhodamine are in common use, but other highly rhodamine to another antibody, one can, for example, 
fluorescent substances are also routinely used, such as phyco- visualize simultaneously two different cell-membrane 
erythrin, an intensely colored and highly fluorescent pig- antigens on the same cell, 
ment obtained from algae. These molecules 
conjugated to the Fc region of an antibody molecule without 



Phycoerythrin is an efficient absorber of light (~30-fold 



greater than fluorescein) and a brilliant emitter of red 
affecting the specificity ot the antibody. Each ot the fluo- ° ...... , . , r 

, ,,,,,., , , , . fluorescence, stimulating its wide use as a label for 

rochromes below absorbs light at one wavelength and emits 

light at a longer wavelength: 



immunofluorescence. 



. . ... , Fluorescent-antibody stamina of cell membrane mole- 

Fluorescein, an organic dye that is the most widely used . . . , ,. . ,. /T ,. „ , , N 

. . , r _ . iiii cules or tissue sections can be direct or indirect (Figure 6- 14). 

label tor immunofluorescence procedures, absorbs blue T ,. . . . .„ ., . . , 

.. . ,,„„ . . . „ In direct staining, the specific antibody (the prima: 

light (490 nm) and emits an intense yellow-green . ... ,. , . , . , „ .. .,. 

_ ,_,„ . body) is directly conjugated with fluorescein; in indirect 

tlnnrpsrpnrp ( SI 7 nm I ' ' ' a 



fluorescence (517 nm). 

Rhodamine, another or 

yellow-green range (515 nm) and emits a deep red ber of reagents have been developed for indirect staining. 



staining, the primary antibody is unlabeled and is detected 
Rhodamine, another organic dye, absorbs in the with an additional fluorochrome-labeled r 



Cells with membrane // lj Primary antibody 

antigens (mAg) /// //L / to mk & 



i h, 



If 



Primary X^^,//^ Secondary 

antibody~~ "-\\ \\ anti-isotype \^ 



V 



U U anti-isotype V W V 4 

£» ^ , antibody , *^S «^S , , ?^5_ 



Fl Fl'i 1 F1 



i 



(a) Direct method with fluorochrome- (b) Indirect method with fluorochrome- (c) Indirec iuorochrome- 

labeled antibody to mAg labeled anti-isotype antibody labeled protein A 

l^iy^^Q Direct and indirect im 

of membrane antigen (mAg). Cells a 

slide. In the direct method (a), cells are 

body that is labeled with a fluorochron 

ods (b and c), cells are first incubate 

antibody and then stained with a fluor 

reagent that binds to the primary antibody. Cells are viewed under 

a fluorescence microscope to see if they have been stained, (d) In 

this micrograph, antibody molecules bearing jul heavy chains are 

detected by indirect staining of cells with rhodamine-conjugated 

second antibody. [Part(d), H. A. Schreuder et al., 1997, Nature 

386:196, courtesy H. Schreuder, Hoechst Marion Roussel.] 



affixed to a n 




.cope 


tained 


with ant 


mA 




(Fl).ln 


theind 


rect 


meth- 


with u 


nlabelec 


ant 


-inAc. 


hrome 


-labeled 




ndary 




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' Immunology 5e: 



ofB-CellandT-Cell Respons 



The most common is a fluorochrome-labeled secondary 
antibody raised in one species against antibodies of another 
species, such as fluorescein-labeled goat anti-mouse im- 
munoglobulin. 

Indirect immunofluorescence staining has two advan- 
tages over direct staining. First, the primary antibody does 
not need to be conjugated with a fluorochrome. Because the 
supply of primary antibody is often a limiting factor, indirect 
methods avoid the loss of antibody that usually occurs dur- 
ing the conjugation reaction. Second, indirect methods in- 
crease the sensitivity of staining because multiple molecules 
of the fluorochrome reagent bind to each primary antibody 
molecule, increasing the amount of light emitted at the loca- 
tion of each primary antibody molecule. 

Immunofluorescence has been applied to identify a num- 
ber of subpopulations of lymphocytes, notably the CD4 + 
and CD8 + T-cell subpopulations. The technique is also suit- 
able for identifying bacterial species, detecting Ag-Ab com- 
plexes in autoimmune disease, detecting complement 
components in tissues, and localizing hormones and other 
cellular products stained in situ. Indeed, a major application 
of the fluorescent-antibody technique is the localization of 
antigens in tissue sections or in subcellular compartments. 
Because it can be used to map the actual location of target 
antigens, fluorescence microscopy is a powerful tool for relat- 
ing the molecular architecture of tissues and organs to their 
overall gross anatomy. 



Flow Cytometry and Fluorescence 

The fluorescent antibody techniques described are ex- 
tremely valuable qualitative tools, but they do not give 
quantitative data. This shortcoming was remedied by 
development of the flow cytometer, which was designed to 
automate the analysis and separation of cells stained with 
fluorescent antibody. The flow cytometer uses a laser beam 
and light detector to count single intact cells in suspension 
(Figure 6-15). Every time a cell passes the laser beam, light is 
deflected from the detector, and this interruption of the 
laser signal is recorded. Those cells having a fluorescently 
tagged antibody bound to their cell surface antigens are ex- 
cited by the laser and emit light that is recorded by a second 
detector system located at a right angle to the laser beam. 
The simplest form of the instrument counts each cell as it 
passes the laser beam and records the level of fluorescence 
the cell emits; an attached computer generates plots of the 
number of cells as the ordinate and their fluorescence inten- 
sity as the abscissa. More sophisticated versions of the in- 
strument are capable of sorting populations of cells into 
different containers according to their fluorescence profile. 
Use of the instrument to determine which and how many 
members of a cell population bind fluorescently labeled an- 
tibodies is called analysis; use of the instrument to place cells 
having different patterns of reactivity into different c 
ers is called cell sorting. 



The flow cytometer has multiple applications to clinical 
and research problems. A common clinical use is to deter- 
mine the kind and number of white blood cells in blood 
samples. By treating appropriately processed blood sam- 
ples with a fluorescently labeled antibody and performing 
flow cytometric analysis, one can obtain the following 
information: 

■ How many cells express the target antigen as an absolute 
number and also as a percentage of cells passing the 
beam. For example, if one uses a fluorescent antibody 
specific for an antigen present on all T cells, it would be 
possible to determine the percentage of T cells in the 
total white blood cell population. Then, using the 
cell-sorting capabilities of the flow cytometer, it would 
be possible to isolate the T-cell fraction of the leukocyte 
population. 

■ The distribution of cells in a sample population 
according to antigen densities as determined by 
fluorescence intensity. It is thus possible to obtain a 
measure of the distribution of antigen density within the 
population of cells that possess the antigen. This is a 
powerful feature of the instrument, since the same type 
of cell may express different levels of antigen depending 
upon its developmental or physiological state. 

■ The size of cells. This information is derived from 
analysis of the light-scattering properties of members of 
the cell population under examination. 

Flow cytometry also makes it possible to analyze cell pop- 
ulations that have been labeled with two or even three differ- 
ent fluorescent antibodies. For example, if a blood sample is 
reacted with a fluorescein-tagged antibody specific for T 
cells, and also with a phycoerythrin-tagged antibody specific 
for B cells, the percentages of B and T cells may be deter- 
mined simultaneously with a single analysis. Numerous vari- 
ations of such "two-color" analyses are routinely carried out, 
and "three-color" experiments are common. Aided by appro- 
priate software, highly sophisticated versions of the flow cy- 
tometer can even perform "five-color" analyses. 

Flow cytometry now occupies a key position in im- 
munology and cell biology, and it has become an indispens- 
able clinical tool as well. In many medical centers, the flow 
cytometer is one of the essential tools for the detection and 
classification of leukemias (see the Clinical Focus). The 
choice of treatment for leukemia depends heavily on the cell 
types involved, making precise identification of the neoplas- 
tic cells an essential part of clinical practice. Likewise, the 
rapid measurement of T-cell subpopulations, an important 
prognostic indicator in AIDS, is routinely done by flow- 
cytometric analysis. In this procedure, labeled monoclonal 
antibodies against the major T-cell subtypes bearing the 
CD4 and CD8 antigens are used to determine their ratios in 
the patient's blood. When the number of CD4 T cells falls 
below a certain level, the patient is at high risk for oppor- 
nfections. 



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;: Principles and Application; 



Ultrasonic 

nozzle vibrator . L J 



5 and 




A - B - 


" cells 




cclls 


on of flu 


orochro 


ne-labeled eel 



Cells stained with: 





Q Oi o ~* 






A- B+ cells 


A+ B+ cells 


A- B- cells 


A+ B- cells 



k antibody fluorescence - 



with the 

flow cytometer. In the example shown, a mixed cell population is 
stained with two antibodies, one specific for surface antigen A and 
the other specific for surface antigen B. The anti-A antibodies are 
labeled with fluorescein (green) and the anti-B antibodies with rho- 
damine (red). The stained cells are loaded into the sample cham- 
ber of the cytometer. The cells are expelled, one at a time, from a 
small vibrating nozzle that generates microdroplets, each contain- 
ing no more than a single cell. As it leaves the nozzle, each droplet 

the flow cytometer can detect exactly when a drop generated by the 
nozzle passes through the beam of laser light that excites the fluo- 
rochrome. The intensity of the fluorescence emitted by each 
droplet that contains a cell is monitored by a detector and dis- 
played on a computer screen. Because the computer tracks the 
position of each droplet, it is possible to determine when a partic- 



ular droplet will arrive between the deflection plates. By applying a 
momentary charge to the deflection plates when a droplet is pass- 
ing between them, it is possible to deflect the path of a particular 
droplet into one or another collecting vessel. This allows the sort- 
ing of a population of cells into subpopulations having different 
profiles of surface markers. 

In the computer display, each dot represents a cell. Cells that fall 
into the lower left-hand panel have background levels of fluorescence 
and are judged not to have reacted with either antibody anti-A or anti-B. 
Those that appear in the upper left panel reacted with anti-B but not 
anti-A, and those in the lower right panel reacted with anti-A but not 
anti-B. The upper right panel contains cells that react with both anti-A 
and anti-B. In the example shown here, the A~B~— and the A + B + — 
subpopulations have each been sorted into a separate tube. Staining 
with anti-A and anti-B fluorescent antibodies allows four subpopula- 
tions to be distinguished: A~B~, A + B + , A~B + , and A + B~. 



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ofB-CeilandT-Cell Respons 



CLINICAL FOCUS 



Flow Cytometry and 
Leukemia Typing 



Leukemi 



; the 



checked proliferation of an abnormal 
clone of hematopoietic cells. Typically, 
leukemic cells respond poorly or inap- 
propriately to regulatory signals, display 
aberrant patterns of differentiation, or 
even fail to differentiate. Furthermore, 
they sometimes suppress the growth of 
normal lymphoid and myeloid cells. 
Leukemia can arise at any maturational 
stage of any one of the hematopoietic 
lineages. Lymphocytic leukemias display 
many characteristics of cells of the lym- 
phoid lineage; another broad group, 
myelogenous leukemias, have attributes 
of members of the myeloid lineage. 
Aside from lineage, many leukemias 
can be classified as acute or chronic. 
Some examples are acute lymphocytic 
leukemia (ALL), the most common 
childhood leukemia; acute myelogenous 
leukemia (AML), found more often in 



adults than in children; and chronic lym- 
phocytic leukemia (CLL), which is rarely 
seen in children but is the most com- 
mon form of adult leukemia in the 
Western world. A fourth type, chronic 
myelogenous leukemia (CML), occurs 
much more often in older adults than in 
children. 

The diagnosis of leukemia is made 
on the basis of two findings. One is the 
detection of abnormal cells in the blood- 
stream, and the other is observation of 
abnormal cells in the bone marrow. Clin- 
ical experience has shown that designing 
the most appropriate therapy for the pa- 
tient requires knowing which type of 
leukemia is present. In this regard, two 
of the important questions are: (1) What 
is the lineage of the abnormal cells and 
(2) What is their maturational stage? A 
variety of approaches, including cyto- 
logic examination of cell morphology 
and staining characteristics, immuno- 



phenotyping, and, in some cases, an 
analysis of gene rearrangements, are 
useful in answering these questions. 
One of the most powerful of these ap- 
proaches is immunophenotyping, the 
determination of the profile of selected 
cell-surface markers displayed by the 
leukemic cell. Although leukemia-spe- 
cific antigens have not yet been found, 
profiles of expressed surface antigens of- 
ten can establish cell lineage, and they 
are frequently helpful in determining the 
maturational stages present in leukemic 
cell populations. For example, an abnor- 
mal cell that displays surface immuno- 
globulin would be assigned to the B-cell 
lineage and its maturational stage would 
be that of a mature B cell. On the other 
hand, a cell that had cytoplasmic |jl heavy 
chains, but no surface immuno-globulin, 
would be a B-lineage leukemic cell but at 
the maturational stage of a pre-B cell. 
The most efficient and precise technol- 
ogy for immunophenotyping uses flow 
cytometry and monoclonal antibodies. 
The availaDinty of monoclonal antibod- 
ies specific for each of the scores of anti- 
gens found on various types and sub- 
types of hematopoietic cells has made it 
possible to identify patterns of antigen 



Alternatives to Antigen-Antibody 
Reactions 

As a defense against host antibodies, some bacteria have 
evolved the ability to make proteins that bind to the Fc region 
of IgG molecules with high affinity (X a ~ 10 8 ). One such 
molecule, known as protein A, is found in the cell walls of 
some strains of Staphylococcus aureus, and another, protein 
G, appears in the walls of group C and G Streptococcus. By 
cloning the genes for protein A and protein G and generating 
a hybrid of both, one can make a recombinant protein, 
known as protein A/G, that combines some of the best fea- 
tures of both. These molecules are useful because they bind 
IgG from many different species. Thus they can be labeled 
with flourochromes, radioactivity, or biotin and used to de- 
tect IgG molecules in the antigen-antibody complexes 
formed during ELISA, RIA, or such fluorescence-based as- 
says as flow cytometry or fluorescence microscopy. These 
bacterial IgG-binding proteins can also be used to make 
affinity columns for the isolation of IgG. 



Egg whites contain a protein called avidin that binds biotin, 
a vitamin that is essential for fat synthesis. Avidin is believed to 
have evolved as a defense against marauding rodents that rob 
nests and eat the stolen eggs. The binding between avidin and 
biotin is extremely specific and of much higher affinity (K a ~ 
10 15 ) than any known antigen-antibody reaction. A bacterial 
protein called streptavidin, made by streptomyces avidinii, has 
similarly high affinity and specificity. The extraordinary affin- 
ity and exquisite specificity of the interaction of these proteins 
with biotin is widely used in many immunological procedures. 
The primary or secondary antibody is labeled with biotin and 
allowed to react with the target antigen, and the unbound an- 
tibody is then washed away. Subsequently, streptavidin or 
avidin conjugated with an enzyme, flourochrome, or radioac- 
tive label is used to detect the bound antibody. 



Immunoelectron Microscopy 

The fine specificity of antibodies has made them powerful 
tools for visualizing specific intracellular tissue components 



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8536d_ch06_137-160 8/1/02 12:41PM Page 157 n 



;: Principles and Application; 



expression that are typical of cell lin- 
eages, maturational stages, and a num- 
ber of different types of leukemia. Most 
cancer centers are equipped with flow cy- 
tometers that are capable of performing 
and interpreting the multiparameter 
analyses necessary to provide useful pro- 



files of surface markers on tumor cell patterns of cytochemical staining 



populations. Flow cytometric determin 
tion of immuno-phenotypes allows: 

Confirmation of diagnosis 
Diagnosis when no clear judgmen 
can be made based on morphology o 



Identification of aberrant antigen pro- 
files that can help identify the return of 
leukemia during remission 
Improved prediction of the course of 
the disease 



n ALL of the pre-B lineage 

>t commonly occurring ALL) 



ALL of the T lineage 




(B-cell marker) 



typical surface 



by immunoelectron microscopy. In this technique, an elec- 
tron-dense label is either conjugated to the Fc portion of a 
specific antibody for direct staining or conjugated to an anti- 
immunoglobulin reagent for indirect staining. A number of 
electron-dense labels have been employed, including ferritin 
and colloidal gold. Because the electron-dense label absorbs 
electrons, it can be visualized with the electron microscope 
as small black dots. In the case of immunogold labeling, dif- 
ferent antibodies can be conjugated with gold particles of 
different sizes, allowing identification of several antigens 
within a cell by the different sizes of the electron-dense gold 
particles attached to the antibodies (Figure 6-16). 



An immunoelectronmicrograph of the surface of a 
a was stained with two antibodies: one against class 
labeled with 30-nm gold particles, and another 

; I molecules labeled with 15-nm gold particles. 

5 I molecules exceeds that of class II on this cell. 

m A.Jenei et al., 1997, PNAS 94:7269-7274; cour- 

S. Damjanovich, University Medical School ofDe- 



B-cell lymphoma v 
II MHC molecule; 
against MHC clas 
The density of clas 
Bar = 500nm./rr< 
tesy of A.Jenei and 
brecen, Hungary.] 




!* ■ 



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8536d_ch06_137-160 8/1/02 12:41PM Page 158 



ofB-CeilandT-Cell Respons 



■ Antigen-antibody interactions depend on four types 
of noncovalent interactions: hydrogen bonds, ionic 
bonds, hydrophobic interactions, and van der Waals 
interactions. 

■ The affinity constant, which can be determined by 
Scatchard analysis, provides a quantitative measure of the 
strength of the interaction between an epitope of the anti- 
gen and a single binding site of an antibody. The avidity re- 
flects the overall strength of the interactions between a 
multivalent antibody molecule and a multivalent antigen 
molecule at multiple sites. 

■ The interaction of a soluble antigen and precipitating anti- 
body in a liquid or gel medium forms an Ag-Ab precipi- 
tate. Electrophoresis can be combined with precipitation 
in gels in a technique called immunoelectrophoresis. 

■ The interaction between a particulate antigen and aggluti- 
nating antibody (agglutinin) produces visible clumping, or 
agglutination that forms the basis of simple, rapid, and 
sensitive immunoassays. 

■ Radioimmunoassay (RIA) is a highly sensitive and quanti- 
tative procedure that utilizes radioactively labeled antigen 
or antibody. 

■ The enzyme-linked immunosorbent assay (ELISA) de- 
pends on an enzyme-substrate reaction that generates a 
colored reaction product. ELISA assays that employ 
chemiluminescence instead of a chromogenic reaction are 
the most sensitive immunoassays available. 

■ In Western blotting, a protein mixture is separated by elec- 
trophoresis; then the protein bands are electrophoretically 
transferred onto nitrocellulose and identified with labeled 
antibody or labeled antigen. 

■ Fluorescence microscopy using antibodies labeled with 
fluorescent molecules can be used to visualize antigen on 
or within cells. 

■ Flow cytometry provides an unusually powerful technol- 
ogy for the quantitative analysis and sorting of cell popula- 
tions labeled with one or more fluorescent antibodies. 



References 

Berzofsky, J. A., I. J. Berkower, and S. L. Epstein. 1991. Antigen- 
antibody interactions and monoclonal antibodies. In Funda- 
mental Immunology, 3rd ed., W. E. Paul, ed. Raven Press, New 
York. 

Coligan, J. E., A. M. Kruisbeek, D. H. Margulies, E. M. Shevach, 
and W. Strober. 1997. Current Protocols in Immunology. Wiley, 
New York. 

Harlow, E., and D. Lane. 1999. Using Antibodies: A laboratory 
manual. Cold Spring Harbor Laboratory Press. 

Herzenberg, L. A., ed. 1996. Weir's Handbook of Experimental 
Immunology, 5th ed. Oxford, Blackwell Scientific Publications. 



Rose, N. R., E. C. de Macario, J. D. Folds, C. H. Lane, and R. M. 
Nakamura. 1997. Manual of Clinical Laboratory Immunology. 
American Society of Microbiology, Washington, D.C. 

Stites, D. P., C. Rodgers, J. D. Folds, and J. Schmitz. 1997. Clinical 
laboratory detection of antigens and antibodies. In Medical 
Immunology, 9th ed., D. P. Stites, A. I. Terr, and T. G. Parslow, 
eds., Appelton and Lange, Stamford, CT. 

Wild, D.,ed. 2001. Their indbook. Nature Publish- 

ing Group, NY. 



SEFUL WEB SITES 



http://pathlabsofark.com/flowcyttests.html 

Explore the Pathology Laboratories of Arkansas to see what 
kinds of samples are taken from patients and what markers 
are used to evaluate lymphocyte populations by flow cy- 
tometry. 

http://jcsmr.anu.edu.au/facslab/AFCG/standards.html 

At the highly informative Australian Flow Cytometry Group 
Web site, one can find a carefully detailed and illustrated 
guide to the interpretation of flow cytometric analyses of clin- 
ical samples. 

http://www.kpl.com 

The Kirkegaard & Perry Laboratories Web site contains a sub- 
site, http://www.kpl.com/support/immun/pds/50datasht/54- 
12-10.html, which allows one to follow a step-by-step 
procedure for using a chemiluminescent substrate in a sensi- 
tive immunoassay. 



Study Questions 



Clinical Focus Question Flow-cytometric analysis for the de- 
tection and measurement of subpopulations of leukocytes, in- 
cluding those of leukemia, is usually performed using mono- 
clonal antibodies. Why is this the case? 



1 . Indicate whether each of the following statements is true or 
false. If you think a statement is false, explain why. 

a. Indirect immunofluorescence is a more sensitive tech- 
nique than direct immunofluorescence. 

b. Most antigens induce a polyclonal response. 

c. A papain digest of anti-SRBC antibodies can agglutinate 
sheep red blood cells (SRBCs). 

d. A pepsin digest of anti-SRBC antibodies can agglutinate 
SRBCs. 

e. Indirect immunofluorescence can be performed using a 
Fab fragment as the primary, nonlabeled antibody. 

f. For precipitation to occur, both antigen and antibody 
must be multivalent. 

g. Analysis of a cell population by flow cytometry can 
simultaneously provide information on both the size 
distribution and antigen profile of cell populations 
containing several different cell types. 



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' Immunology 5e: 



;: Principles and Application; 



h. ELISA tests using chemiluminescence are more sensitive 
than chromogenic ones and precipitation tests are more 
sensitive than agglutination tests. 

i. Western blotting and immunoprecipitation assays are 
useful quantitative assays for measuring the levels of pro- 

j. Assume antibody A and antibody B both react with an 
epitope C. Furthermore, assume that antibody A has a _fC a 
5 times greater than that of antibody B. The strength of 
the monovalent reaction of antibody A with epitope C 
will always be greater than the avidity of antibody B for 
an antigen with multiple copies of epitope C. 

2. You have obtained a preparation of purified bovine serum 
albumin (BSA) from normal bovine serum. To determine 
whether any other serum proteins remain in this prepara- 
tion of BSA, you decide to use immunoelectrophoresis. 

a. What antigen would you use to prepare the mm 
■''-'-- in the BSA preparation? 



. How could you prodi 
could be used 



isotype-specific antibodies that 
the isotype of myeloma pro- 



needed to detect impurit™ „. 
b. Assuming that the BSA preparation is pure, draw the uu- 
munoelectrophoretic pattern you would expect if the assay 
was performed with bovine serum in a well above a trough 
containing the antiserum you prepared in (a) and the BSA 
sample in a well below the trough as shown below: 




. The labels from four bottles (A, B, C, and D) of hapten- 
carrier conjugates were accidentally removed. However, it 
was known that each bottle contained either 1) hapten 
1-carrier 1 (Hl-Cl), 2) hapten 1-carrier 2 (H1-C2), 
3) hapten 2-carrier 1 (H2-C1), or 4) hapten 2-carrier 
2 (H2-C2). Carrier 1 has a molecular weight of 60,000 dal- 
tons and carrier 2 has a molecular weight of over 120,000 
daltons. Assume you have an anti-Hl antibody and an anti- 
H-2 antibody and a molecular-weight marker that is 100,000 
daltons. Use Western blotting to determine the contents of 
each bottle and show the Western blots you would expect 
from 1, 2, 3, and 4. Your answer should also tell which anti- 
body or combination of antibodies was used to obtain each 
blot. 



n of a small amount (250 nanograms/ml) 
of hapten can be determined by which of the following as- 
says: (a) ELISA (chromogenic), (b) Ouchterlony method, 
(c) RIA, (d) fluorescence microscopy, (e) flow cytometry, 
(f) immunoprecipitation, (g) immunoelectron microscopy, 
(h) ELISPOT assay, (i) chemiluminescent ELISA. 

5. You have a myeloma protein, X, whose isotype is unknown 
and several other myeloma proteins of all known isotypes 
(e.g.,IgG,IgM,IgA,andIgE). 



b. How could you us> 
the level of myelo: 



:his anti-isotype antibody ti 
i protein X in normal serur 



6. For each antigen or antibody listed below, indicate an appro- 
priate assay method and the necessary test reagents. Keep in 
mind the sensitivity of the assay and the expected concentra- 
tion of each protein. 

a. IgG in serum 

c. IgE in serum 

d. Complement component C3 on glomerular basement 

e. Anti-A antibodies to blood-group antigen A in serum 

f. Horsemeat contamination of hamburger 



r from a char 

ipate in the formation 



g. Syphilis spirochet 

7. Which of the following does ; 
of antigen-antibody complex 

a. Hydrophobic bonds 

b. Covalent bonds 

d. Hydrogen bonds 

e. Van der Waals forces 



8. Explain the difference between antibody affinity and anti- 
body avidity. Which of these properties of an antibody better 
reflects its ability to contribute to the humoral immune re- 
sponse to invading bacteria? 

9. You want to develop a sensitive immunoassay for a hor- 
mone that occurs in the blood at concentrations near 
10~ 7 M. You are offered a choice of three different antisera 
whose affinities for the hormone have been determined by 
equilibrium dialysis. The results are shown in the Scatchard 




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' Immunology 5e: 



ofB-CeilandT-Cell Respons 



a. What is the value of K for each a: 

b. What is the valence of each of the antibodies? 

c. Which of the antisera might be a monoclonal antibody? 

d. Which of the antisera would you use for your assay? 
Why? 

I. In preparing a demonstration for her immunology class, 
an instructor purified IgG antibodies to sheep red blood 
cells (SRBCs) and digested some of the antibodies into 
Fab, Fc, and F(ab.)2 fragments. She placed each prepara- 
tion in a separate tube, labeled the tubes with a water- 
soluble marker, and left them in an ice bucket. When the 
instructor returned for her class period, she discovered 
that the labels had smeared and were unreadable. Deter- 
mined to salvage the demonstration, she relabeled the 
tubes 1, 2, 3, and 4 and proceeded. Based on the test results 
described below, indicate which preparation was con- 
tained in each tube and explain how you identified the 



a. The preparation in tube 1 agglutinated SRBCs but did 
not lyse them in the presence of complement. 

b. The preparation in tube 2 did not agglutinate SRBCs or 
lyse them in the presence of complement. However, when 
this preparation was added to SRBCs before the addition 
of whole anti-SRBC, it prevented agglutination of the 
cells by the whole anti-SRBC antiserum. 

c. The preparation in tube 3 agglutinated SRBCs and also 
lysed the cells in the presence of complement. 



d. The preparati 
BCsanddidi 
anti-SRBC 



in tube 4 did not agglutinate or lyse SR- 
nhibit agglutination of SRBCs by whole 



1 1 . You are given two solutions, one containing protein X and 
the other containing antibody to protein X. When you add 1 
ml of anti-X to 1 ml of protein X, a precipitate forms. But 
when you dilute the antibody solution 100-fold and then 
mix 1 ml of the diluted anti-X with 1 ml of protein X, no pre- 
cipitate forms. 



pirate rorms. 

Explain why no precipitate formed with the diluted anti- 
lld likely be pre- 



b. Which species (protein X or anti-X) would lik 
sent in the supernatant of the antibody-antige 



12. Consider equation 1 and derive the form of the Scatchard 
equation that appears in equation 2. 



1. S + L = SL 

2. B/F = Ka([S], ■ 






Where: S = antibody binding sites; [S] = molar c 
of antibody binding sites; L = ligand (monovalent antigen); 
[L] = molar concentration of ligand; SL = site-ligand complex; 
[SL] = molar concentration of site ligand complex; B is substi- 
tuted for [SL] and F for [L]. Hint: It will be helpful to begin by 
writing the law of mass action for the reaction shown in equa- 



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8536d_ch07_161-184 8/15/02 



:114 Mac 114:2nd^ 






Major 

Histocompatibility 

Complex 



rVERY MAMMALIAN SPECIES STUDIED TO DATE 
possesses a tightly linked cluster of genes, the ma- 
jor histocompatibility complex (MHC), whose 
s play roles in intercellular recognition and in dis- 
ween self and nonself. The MHC partici- 
pates in the development of both humoral and cell- 
mediated immune responses. While antibodies may react 
with antigens alone, most T cells recognize antigen only 
when it is combined with an MHC molecule. Furthermore, 
because MHC molecules act as antigen-presenting struc- 
tures, the particular set of MHC molecules expressed by an 
individual influences the repertoire of antigens to which that 
individual's T H and T c cells can respond. For this reason, the 
MHC partly determines the response of an individual to 
antigens of infectious organisms, and it has therefore been 
implicated in the susceptibility to disease and in the devel- 
opment of autoimmunity. The recent understanding that 
natural killer cells express receptors for MHC class I antigens 
and the fact that the receptor-MHC interaction may lead to 
inhibition or activation expands the known role of this gene 
family (see Chapter 14). The present chapter examines the 
organization and inheritance of MHC genes, the structure of 
the MHC molecules, and the central function that these 
molecules play in producing an immune response. 



General Organization and 
Inheritance of the MHC 

The concept that the rejection of foreign tissue is the result 
of an immune response to cell-surface molecules, now called 
histocompatibility antigens, originated from the work of 
Peter Gorer in the mid- 1930s. Gorer was using inbred strains 
of mice to identify blood-group antigens. In the course of 
these studies, he identified four groups of genes, designated 
I through IV, that encoded blood-cell antigens. Work carried 
out in the 1940s and 1950s by Gorer and George Snell estab- 
lished that antigens encoded by the genes in the group desig- 
nated II took part in the rejection of transplanted tumors 
and other tissue. Snell called these genes "histocompatibility 



char. 


)ter 7 




1 V 






51 

l v 




n 


Presentation of Vesicular Stomatitis Virus Peptide (top) 
and Sendai Virus Nucleoprotein Peptide by Mouse MHC 
Class 1 Molecule H-2K b 



■ General Organization and Inheritance of the MHC 

■ MHC Molecules and Genes 

■ Detailed Genomic Map of MHC Genes 

■ Cellular Distribution of MHC Molecules 

■ Regulation of MHC Expression 

■ MHC and Immune Responsiveness 

■ MHC and Disease Susceptibility 



genes"; their current designation as histocompatibility-2 
(H-2) genes was in reference to Gorer's group II blood-group 
antigens. Although Gorer died before his contributions were 
recognized fully, Snell was awarded the Nobel prize in 1980 
for this work. 

The MHC Encodes Three Major 
Classes of Molecules 

The major histocompatibility complex is a collection of 
genes arrayed within a long continuous stretch of DNA on 
chromosome 6 in humans and on chromosome 17 in mice. 
The MHC is referred to as the HLA complex in humans and 
as the H-2 complex in mice. Although the arrangement of 
genes is somewhat different, in both cases the MHC genes are 
organized into regions encoding three classes of molecules 
(Figure 7-1): 

■ Class I MHC genes encode glycoproteins expressed on 
the surface of nearly all nucleated cells; the major 
function of the class I gene products is presentation of 
peptide antigens to T c cells. 



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8536d:Goldsby et al . / Immunology 5e-: 



Generation of B-Cell and T-Cell Respor 



VISUALIZING CONCEPTS 

Mouse H-2 complex 



Complex 


H-2 


MHC class 


I 


II 


III 


I 


Region 


K 


IA 


IE 


s 


D 


Gene 


H-2K 


IA 

ap 


IE 

«[3 


C' proteins 


TNF-a 
TNF-P 


H-2D 


H-2L 



Complex 


HLA 


MHC class 


n 


III 


I 


Region 


DP 


DQ 


DR 


C4, C2, BF 


B 


C 


A 


Gene 
products 


DP 

cxp 


DQ 

ap 


DR 

ap 


C' proteins 


TNF-a 
TNF-P 


HLA-B 


HLA-C 


HLA-A 



ibility complex (MHC) in the mouse 
ferred to as the H-2 complex in mice 
humans. In both species the MHC is 



jman.TheMHCis 
s the HLA complex 



n) gene products. The class I and class II gene products 
n in this figure are considered to be the classical MHC mol- 
s. The class III gene products include complement (C) pro- 
is factors (TNF-a and TNF-P). 



■ Class II MHC genes encode glycoproteins expressed 
primarily on antigen-presenting cells (macrophages, 
dendritic cells, and B cells), where they present processed 
antigenic peptides to T H cells. 

■ Class III MHC genes encode, in addition to other 
products, various secreted proteins that have immune 
functions, including components of the complement 
system and molecules involved in inflammation. 

Class I MHC molecules encoded by the K and D regions in 
mice and by the A, B, and C loci in humans were the first 
discovered, and they are expressed in the widest range of 
cell types. These are referred to as classical class I molecules. 
Additional genes or groups of genes within the H-2 or HLA 
complexes also encode class I molecules; these genes are 
designated nonclassical class I genes. Expression of the non- 
classical gene products is limited to certain specific cell 
types. Although functions are not known for all of these 
gene products, some may have highly specialized roles in 
immunity. For example, the expression of the class I HLA- 
G molecules on cytotrophoblasts at the fetal-maternal in- 
terface has been implicated in protection of the fetus from 
being recognized as foreign (this may occur when paternal 



antigens begin to appear) and from being rejected by ma- 
ternal T c cells. 

The two chains of the class II MHC molecules are en- 
coded by the IA and IE regions in mice and by the DP, DQ, 
and DR regions in humans. The terminology is somewhat 
confusing, since the D region in mice encodes class I MHC 
molecules, whereas the D region (DR, DQ, DP) in humans 
refers to genes encoding class II MHC molecules! Fortu- 
nately, the designation D for the general chromosomal loca- 
tion encoding the human class II molecules is seldom used 
today; the sequence of the entire MHC region is available so 
the more imprecise reference to region is seldom necessary. 
As with the class I loci, additional class II molecules en- 
coded within this region have specialized functions in the 
immune process. 

The class I and class II MHC molecules have common 
structural features and both have roles in antigen processing. 
By contrast, the class III MHC region, which is flanked by the 
class I and II regions, encodes molecules that are critical to 
immune function but have little in common with class I or II 
molecules. Class III products include the complement com- 
ponents C4, C2, BF (see Chapter 13), and inflammatory cy- 
tokines, including tumor necrosis factor (TNF) and 
heat-shock proteins (see Chapter 12). 



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:853 6d:Goldsby e 



Major Histocompatibility Complex 



Allelic Forms of MHC Genes Are Inherited 
in Linked Groups Called Haplotypes 

As described in more detail later, the loci constituting the 
MHC are highly polymorphic; that is, many alternative 
forms of the gene, or alleles, exist at each locus among the 
population. The genes of the MHC loci lie close together; for 
example, the recombination frequency within the H-2 com- 
plex (i.e., the frequency of chromosome crossover events 
during mitosis, indicative of the distance between given gene 
segments) is only 0.5% — crossover occurs only once in every 
200 mitotic cycles. For this reason, most individuals inherit 
the alleles encoded by these closely linked loci as two sets, one 
from each parent. Each set of alleles is referred to as a haplo- 
type. An individual inherits one haplotype from the mother 
and one haplotype from the father. In outbred populations, 
the offspring are generally heterozygous at many loci and will 
express both maternal and paternal MHC alleles. The alleles 
are codominantly expressed; that is, both maternal and pater- 
nal gene products are expressed in the same cells. If mice are 
inbred (that is, have identical alleles at all loci), each H-2 lo- 
cus will be homozygous because the maternal and paternal 
haplotypes are identical, and all offspring therefore express 
identical haplotypes. 

Certain inbred mouse strains have been designated as 
prototype strains, and the MHC haplotype expressed by 
these strains is designated by an arbitrary italic superscript 
(e.g., H-2 a , H-2 6 ). These designations refer to the entire set of 
inherited H-2 alleles within a strain without having to list 
each allele individually (Table 7-1). Different inbred strains 
may have the same set of alleles, that is the same MHC hap- 
lotype, as the prototype strain. For example, the CBA, AKR, 
and C3H strains all have the same MHC haplotype (H-2*). 
The three strains differ, however, in genes outside the H-2 
complex. 

If two mice from inbred strains having different MHC 
haplotypes are bred to one another, the Fj generation inher- 
its haplotypes from both parental strains and therefore ex- 



presses both parental alleles at each MHC locus. For exam- 
ple, if an H-2 b strain is crossed with an H-2*, then the F 1 in- 
herits both parental sets of alleles and is said to be H-2 ' 
(Figure 7-2a). Because such an F! expresses the MHC pro- 
teins of both parental strains on its cells, it is histocompatible 
with both strains and able to accept grafts from either 
parental strain (see example in Figure 7-2b). However, nei- 
ther of the inbred parental strains can accept a graft from the 
F[ mice because half of the MHC molecules will be foreign to 
the parent. 

The inheritance of HLA haplotypes from heterozygous 
human parents is illustrated in Figure 7-2c. In an outbred 
population, each individual is generally heterozygous at each 
locus. The human HLA complex is highly polymorphic and 
multiple alleles of each class I and class II gene exist. How- 
ever, as with mice, the human MHC loci are closely linked 
and usually inherited as a haplotype. When the father and 
mother have different haplotypes, as in the example shown 
(Figure 7-2c) there is a one-in-four chance that siblings will 
inherit the same paternal and maternal haplotypes and 
therefore be histocompatible with each other; none of the 
offspring will be histocompatible with the parents. 

Although the rate of recombination by crossover is low 
within the HLA, it still contributes significantly to the diver- 
sity of the loci in human populations. Genetic recombina- 
tion generates new allelic combinations (Figure 7-2d), and 
the high number of intervening generations since the ap- 
pearance of humans as a species has allowed extensive re- 
combination, so that it is rare for any two unrelated 
individuals to have identical sets of HLA genes. 

MHC Congenic Mouse Strains Are Identical 
at All Loci Except the MHC 

Detailed analysis of the H-2 complex in mice was made 
possible by the development of congenic mouse strains. In- 
bred mouse strains are syngeneic or identical at all genetic 
loci. Two strains are congenic if they are genetically identical 



■ 



Prototype strain 


Other strains with the same haplo 


CBA 


AKR, C3H, B10.BR, C57BR 


DBA/2 


BALB/c, NZB, SEA.YBR 


C57BL/10(B10) 


C57BL/6, C57L, C3H.SW, LP, 129 


A 


A/He, A/Sn,A/Wy, B10.A 


ASW 


B10.S, SJL 


ATL 




DBA/1 


STOLI, B10.Q, BDP 



A 



8536d_ch07_161-18. 



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8536d:Goldsby e 



al . / Immunology 5e-: 



(a) Mating of inbred moui iffcrent MHC haplotypes 

Homologous chromosomes with MHC loci 
H-2 6 parent II H-2* parent 

bib klk 



mb it 



F! progeny (H-2 6 / fe ) 



different MHC haplotypes 



Parental recipient 



Progeny recipient 




of MHC haplotypes in inbred mouse strains. 
The letters b/b designate a mouse homozy- 
gous for the H-2 fa MHC haplotype, k/k ho- 
mozygous for the H-2 k haplotype, and b/k a 
heterozygote. Because the MHC loci are 
closely linked and inherited as a set, the 
MHC haplotype of F1 progeny from the mat- 
ing of two different inbred strains can be pre- 
dicted easily, (b) Acceptance or rejection of 
skin grafts is controlled by the MHC type of 
the inbred mice. The progeny of the cross be- 
tween two inbred strains with different MHC 
haplotypes (H-2 b and H-2* c ) will express both 
haplotypes (H-2 b/k ) and will accept grafts 
from either parent and from one another. 
Neither parent strain will accept grafts from 
the offspring, (c) Inheritance of HLA haplo- 
types in a hypothetical human family. In hu- 
mans, the paternal HLA haplotypes are 
arbitrarily designated A and B, maternal C 
and D. Because humans are an outbred 
species and there are many alleles at each 
HLA locus, the alleles comprising the haplo- 
types must be determined by typing parents 
and progeny, (d) The genes that make up 
each parental haplotype in the hypothetical 
family in (c) are shown along with a new hap- 
lotype that arose from recombination (R) of 
maternal haplotypes. 




If^-a-lf^ 



(c) Inheritance of HLA hap; cal human family 

Parents 



II 



1 I 



(d) A new ha 

of maternal haplotypes 









HLA Alleles 






A 


B 


C 


DR 


DQ DP 


A 


1 


7 


w3 


2 


1 1 














13 


2 


8 


W2 


3 


2 2 
















3 


44 


W4 


4 


1 3 | 


D 












R 


3 


44 


W4 







A/C A/D B/R B/C B/D 



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Major Histocompatibility Complex 




Select for b/b 



strain A.B, which has the genetic background of 
parental strain A but the H-2 complex of strain B. 
Crossing inbred strain A (H-2°) with strain B (H-2 b ) 
generates F, progeny that are heterozygous (a/b) 
at all H-2 loci. The F, progeny are interbred to pro- 
duce an F 2 generation, which includes a/a, a/b, 
and b/b individuals. The F 2 progeny homozygous 
for the B-strain H-2 complex are selected by their 
ability to reject a skin graft from strain A; any prog- 
eny that accept an A-strain graft are eliminated 
from future breeding. The selected b/b homozy- 
gous mice are then backcrossed to strain A; the re- 
sulting progeny are again interbred and their 
offspring are again selected for b/b homozygosity 
at the H-2 complex. This process of backcrossing 
to strain A, intercrossing, and selection for ability to 
reject an A-strain graft is repeated for at least 12 
generations. In this way A-strain homozygosity is 
restored at all loci except the H-2 locus, which is 
homozygous for the B strain. 



except at a single genetic locus or region. Any pheno- 
typic differences that can be detected between congenic 
strains are related to the genetic region that distinguishes 
the strains. Congenic strains that are identical with each 
other except at the MHC can be produced by a series of 
crosses, backcrosses, and selections. Figure 7-3 outlines the 
steps by which the H-2 complex of homozygous strain B 
can be introduced into the background genes of homozy- 
gous strain A to generate a congenic strain, denoted A.B. 
The first letter in a congenic strain designation refers to the 
strain providing the genetic background and the second 
letter to the strain providing the genetically different MHC 
region. Thus, strain A.B will be genetically identical to 
strain A except for the MHC locus or loci contributed by 
strain B. 

During production of congenic mouse strains, a crossover 
event sometimes occurs within the H-2 complex, yielding a 
recombinant strain that differs from the parental strains or 
the congenic strain at one or a few loci within the H-2 
complex. Figure 7-4 depicts haplotypes present in several re- 
combinant congenic strains that were obtained during pro- 



A 


a 


1 


BIO 


b 




IS 10. A 


a 


1 








B10.A (2R) 


h2 [_ 


■ 








B10.A (3R) 


(3 | 


1 1 








B10.A (4R) 


h4 [ 


1 1 


B10.A (18R) 


118 ■ 


1 1 



generated during product 
B10 (H-2 b ) and parental s 
H-2 complex produce rec 
alleles (blue) at some H- 
other loci. 



in of the B10.A strain from parental strain 
ain A (H-2°). Crossover events within the 
mbinant strains, which have o-haplotype 
loci and fa-haplotype alleles (orange) at 



duction of a BIO. A congenic strain. Such recombinant 
strains have been extremely useful in analyzing the MHC be- 
cause they permit comparisons of functional differences 



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Generation of B-Cell and T-Cell Respor 



between strains that differ in only a few genes within the 
MHC. Furthermore, the generation of new H-2 haplotypes 
under the experimental conditions of congenic strain devel- 
opment provides an excellent illustration of the means by 
which the MHC continues to maintain heterogeneity even in 
populations with limited diversity. 



MHC Molecules and Genes 

Class I and class II MHC molecules are membrane-bound 
glycoproteins that are closely related in both structure and 
function. Both class I and class II MHC molecules have been 
isolated and purified and the three-dimensional structures 
of their extracellular domains have been determined by x- 
ray crystallography. Both types of membrane glycoproteins 
function as highly specialized antigen-presenting molecules 
that form unusually stable complexes with antigenic pep- 
tides, displaying them on the cell surface for recognition by 
T cells. In contrast, class III MHC molecules are a group of 
unrelated proteins that do not share structural similarity 
and common function with class I and II molecules. The 
class III molecules will be examined in more detail in later 
chapters. 



;s within the 



highly con- 
Association 



Class I Molecules Have a Glycoprotein Heavy 
Chain and a Small Protein Light Chain 

Class I MHC molecules contain a 45-kilodalton (kDa) a 
chain associated noncovalently with a 12-kDa (^-microglob- 
ulin molecule (see Figure 7-5). The a chain is 
brane glycoprotein encoded by polymorphic ger 
A, B, and C regions of the human HLA comple 
the K and D/L regions of the mouse H-2 compl< 
7-1). [^-Microglobulin is a protein encoded by 
served gene located on a different chromosome 
of the a chain with {^-microglobulin is required for expres- 
sion of class I molecules on cell membranes. The a chain is 
anchored in the plasma membrane by its hydrophobic trans- 
membrane segment and hydrophilic cytoplasmic tail. 

Structural analyses have revealed that the a chain of class I 
MHC molecules is organized into three external domains 
(al, a2, and a3), each containing approximately 90 amino 
acids; a transmembrane domain of about 25 hydrophobic 
amino acids followed by a short stretch of charged (hy- 
drophilic) amino acids; and a cytoplasmic anchor segment of 
30 amino acids. The (^-microglobulin is similar in size and 
organization to the a3 domain; it does not contain a trans- 
membrane region and is noncovalently bound to the class I 
glycoprotein. Sequence data reveal homology between the a3 



Class I molecule 



Class II molecule 



(Ig-fold structure) 



Transmembrane segment 





sofa 



cdiagra, 

molecule showing the external domains, t 
and cytoplasmic tail. The peptide-binding cl 
brane-distal domains in both class I and 



s formed by the i 
iss II molecules 



ains possess the basic ii 
and class II MHCmoleci 
loglobulin superfamily. 



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Major Histocompatibility Complex chapter 7 167 





" Representations of the three-dimensional structure of 
the external domains of a human class I MHC molecule based on x- 
ray crystallography analysis, (a) Side view in which the (3 strands are 
depicted as thick arrows and the a helices as spiral ribbons. Disulfide 
bonds are shown as two interconnected spheres. The al and a2 do- 
mains interact to form the peptide-binding cleft. Note the im- 



munoglobulin-fold structure ofthe «3 domain and p 2 -m 


croglobulin. 


(b) The al and a2 domains as viewed from the top, - 


howing the 


peptide-binding cleft consisting of a base of antiparalle 


1 P strands 


and sides of a helices. This cleft in class 1 molecules car 


accommo- 


date peptides containing 8-10 residues. 





domain, (^-microglobulin, and the constant-region domains 
in immunoglobulins. The enzyme papain cleaves the a chain 
just 13 residues proximal to its transmembrane domain, re- 
leasing the extracellular portion ofthe molecule, consisting of 
al, a2, a3, and (^-microglobulin. Purification and crystal- 
lization of the extracellular portion revealed two pairs of in- 
teracting domains: a membrane-distal pair made up of the a 1 
and a2 domains and a membrane-proximal pair composed of 
the a3 domain and ^-microglobulin (Figure 7-6a). 

The al and a2 domains interact to form a platform of 
eight antiparallel (3 strands spanned by two long a-helical re- 
gions. The structure forms a deep groove, or cleft, approxi- 
mately 25 Ax 10AX 11 A, with the long a helices as sides 
and the (3 strands of the (3 sheet as the bottom (Figure 7-6b). 
This peptide-binding cleft is located on the top surface of the 
class I MHC molecule, and it is large enough to bind a peptide 
of 8- 10 amino acids. The great surprise in the x-ray crystallo- 
graphic analysis of class I molecules was the finding of small 
peptides in the cleft that had cocrystallized with the protein. 
These peptides are, in fact, processed antigen and self-pep- 
tides bound to the al and a2 domains in this deep groove. 

The a3 domain and (3 2 -microglobulin are organized into 
two (3 pleated sheets each formed by antiparallel (3 strands of 
amino acids. As described in Chapter 4, this structure, known 
as the immunoglobulin fold, is characteristic of im- 
munoglobulin domains. Because of this structural similarity, 



which is not surprising given the considerable sequence sim- 
ilarity with the immunoglobulin constant regions, class I 
MHC molecules and [3 2 -microglobulin are classified as 
members of the immunoglobulin superfamily (see Figure 
4-20). The a3 domain appears to be highly conserved among 
class I MHC molecules and contains a sequence that interacts 
with the CD8 membrane molecule present on T c cells. 

(3 2 -Microglobulin interacts extensively with the a3 do- 
main and also interacts with amino acids of the al and a2 
domains. The interaction of ^-microglobulin and a peptide 
with a class I a chain is essential for the class I molecule to 
reach its fully folded conformation. As described in detail in 
Chapter 8, assembly of class I molecules is believed to occur 
by the initial interaction of f3 2 -microglobulin with the fold- 
ing class I a chain. This metastable "empty" dimer is then sta- 
bilized by the binding of an appropriate peptide to form the 
native trimeric class I structure consisting of the class I a 
chain, ^-microglobulin, and a peptide. This complete mole- 
cular complex is ultimately transported to the cell surface. 

In the absence of (3 2 -microglobulin, the class I MHC a 
chain is not expressed on the cell membrane. This is illus- 
trated by Daudi tumor cells, which are unable to synthesize 
(3 2 -microglobulin. These tumor cells produce class I MHC a 
chains, but do not express them on the membrane. However, 
if Daudi cells are transfected with a functional gene encoding 
(3 2 -microglobulin, class I molecules appear on the membrane. 



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Class II Molecules Have Two Nonidentical 
Glycoprotein Chains 

Class II MHC molecules contain two different polypeptide 
chains, a 33-kDa a chain and a 28-kDa (3 chain, which asso- 
ciate by noncovalent interactions (see Figure 7-5b). Like class 
I a chains, class II MHC molecules are membrane-bound 
glycoproteins that contain external domains, a transmem- 



anchor segment. Each 

two external domains: 

Ld (31 and (32 domains in 

a2 and (32 domains, like 

croglobulin domains of 



brane segment, and a cytoplas 
chain in a class II molecule o 
al and a2 domains in one chain a 
the other. The membrane-proxima 
the membrane-proximal a3/(3 2 -n 
class I MHC molecules, bear sequence similarity to the im- 
munoglobulin-fold structure; for this reason, class II MHC 
molecules also are classified in the immunoglobulin super- 
family. The membrane-distal portion of a class II molecule is 
composed of the al and (31 domains and forms the antigen- 
binding cleft for processed antigen. 

X-ray crystallographic analysis reveals the similarity of 
class II and class I molecules, strikingly apparent when the 
molecules are surperimposed (Figure 7-7). The peptide- 
binding cleft of HLA-DR1, like that in class I molecules, is 
composed of a floor of eight antiparallel (3 strands and sides 
of antiparallel a helices. However, the class II molecule lacks 
the conserved residues that bind to the terminal residues of 
short peptides and forms instead an open pocket; class I pre- 
sents more of a socket, class II an open-ended groove. These 
functional consequences of these differences in fine structure 
will be explored below. 

An unexpected difference between crystallized class I and 
class II molecules was observed for human DR1 in that the 




ne-distal, peptide-binding cleft of a hu- 
iTmHC molecule, HLA-DR1 (blue), superimposed c 
the corresponding regions of a human class I MHC molecule, HLA- 
A2 (red). [From J. H. Brown et al., 1993, Nature 364:33.] 




(b) fVv 


& 



21 Antigen-binding cleft of dimeric class 1 1 DR1 molecule 
in (a) top view and (b) side view. This molecule crystallized as a 
dimer of the a (3 heterodimer. The crystallized dimer is shown with 
one DR1 molecule in red and the other DR1 molecule in blue. The 
bound peptides are yellow. The two peptide-binding clefts in the 
dimeric molecule face in opposite directions. [From J. H. Brown et al., 
1993, Nature 364:33.] 



latter occurred as a dimer of a (3 heterodimers, a "dimer of 
dimers" (Figure 7-8). The dimer is oriented so that the two 
peptide-binding clefts face in opposite directions. While it has 
not yet been determined whether this dimeric form exists in 
vivo, the presence of CD4 binding sites on opposite sides of 
the class II molecule suggests that it does. These two sites on 
the a2 and (32 domains are adjacent in the dimer form and a 
CD4 molecule binding to them may stabilize class II dimers. 

The Exon/lntron Arrangement of Class I and 
II Genes Reflects Their Domain Structure 

Separate exons encode each region of the class I and II pro- 
teins (Figure 7-9). Each of the mouse and human class I 
genes has a 5' leader exon encoding a short signal peptide 



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Major Histocompatibility Complex chapter 7 169 




?matic diagram of (a) class I and (b) class 
genes, mRNA transcripts, and protein molecules. There i 
spondence between exons and the domains in the gene p 
note that the mRNA transcripts are spliced to remove the ir 
quences. Each exon, with the exception of the leader (L) e 



codes a separate domain of the MHC molecule. The leader peptides 
are removed in a post-translational reaction before the molecules are 
expressed on the cell surface. The gene encoding (3 2 -microglobulin is 
located on a different c 



followed by five or six exons encoding the a chain of the class 
I molecule (see Figure 7-9a). The signal peptide serves to fa- 
cilitate insertion of the a chain into the endoplasmic reticu- 
lum and is removed by proteolytic enzymes in the 
endoplasmic reticulum after translation is completed. The 
next three exons encode the extracellular al, a2, and ct3 do- 
mains, and the following downstream exon encodes the 
transmembrane (T m ) region; finally, one or two 3 '-terminal 
exons encode the cytoplasmic domains (C). 

Like class I MHC genes, the class II genes are organized 
into a series of exons and introns mirroring the domain struc- 
ture of the a and (} chains (see Figure 7-9b). Both the a and 
the p genes encoding mouse and human class II MHC mole- 
cules have a leader exon, an al or (31 exon, an a2 or (32 exon, 
a transmembrane exon, and one or more cytoplasmic exons. 



Class I and II Molecules Exhibit 
Polymorphism in the Region That 
Binds to Peptides 

Several hundred different allelic variants of class I and II MHC 
molecules have been identified in humans. Any one individual, 
however, expresses only a small number of these molecules — 
up to 6 different class I molecules and up to 12 different class II 
molecules. Yet this limited number of MHC molecules must be 
able to present an enormous array of different antigenic pep- 
tides to T cells, permitting the immune system to respond 
specifically to a wide variety of antigenic challenges. Thus, pep- 
tide binding by class I and II molecules does not exhibit the fine 
specificity characteristic of antigen binding by antibodies and 
T-cell receptors. Instead, a given MHC molecule can bind 



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Generation of B-Cell and T-Cell Respor 



| TABLE 7-2 ^ 








■ 




Class 1 molecules 




Class II molecules 




Peptide-binding domain 


«1/a2 




al/B1 




Nature of peptide-binding cleft 


Closed at both ends 




Open at both ends 




General size of bound peptides 


8-10 amino acids 




13-18 amino acids 




Peptide motifs involved in 
binding to MHC molecule 


Anchor residues at both ends 
peptide; generally hydropho 
carboxyl-terminal anchor 


of 


Anchor residues distributed along 
the length of the peptide 




Nature of bound peptide 


Extended structure in which both ends 
interact with MHC cleft but middle 
arches up away from MHC molecule 


Extended structure that is held 
at a constant elevation above 
the floor of MHC cleft 





s different peptides, and some peptides can bind to 
several different MHC molecules. Because of this broad speci- 
ficity, the binding between a peptide and an MHC molecule is 
often referred to as "promiscuous." 

Given the similarities in the structure of the peptide-bind- 
ing cleft in class I and II MHC molecules, it is not surprising 
that they exhibit some common peptide-binding features 
(Table 7-2). In both types of MHC molecules, peptide lig- 
ands are held in a largely extended conformation that runs 
the length of the cleft. The peptide-binding cleft in class I 
molecules is blocked at both ends, whereas the cleft is open in 
class II molecules (Figure 7-10). As a result of this difference, 
class I molecules bind peptides that typically contain 8-10 
amino acid residues, while the open groove of class II mole- 
cules accommodates slightly longer peptides of 13-18 amino 
acids. Another difference, explained in more detail below, is 
that class I binding requires that the peptide contain specific 
amino acid residues near the N and C termini; there is no 
such requirement for class II peptide binding. 

The peptide-MHC molecule association is very stable 
(_K" d ~ 10~ 6 ) under physiologic conditions; thus, most of 



the MHC molecules expressed on the membrane of a 
will be associated with a peptide of self or nonself origir 



CLASS I MHC-PEPTIDE INTERACTION 

Class I MHC molecules bind peptides and present them to 
CD8 + T cells. In general, these peptides are derived from en- 
dogenous intracellular proteins that are digested in the cy- 
tosol. The peptides are then transported from the cytosol 
into the cisternae of the endoplasmic reticulum, where they 
interact with class I MHC molecules. This process, known as 
the cytosolic or endogenous processing pathway, is discussed 
in detail in the next chapter. 

Each type of class I MHC molecule (K, D, and L in mice 
or A, B, and C in humans) binds a unique set of peptides. In 
addition, each allelic variant of a class I MHC molecule (e.g., 
H-2K* and H-2K d ) also binds a distinct set of peptides. Be- 
cause a single nucleated cell expresses about 10 5 copies of 
each class I molecule, many different peptides will be ex- 
pressed simultaneously on the surface of a nucleated cell by 
class I MHC molecules. 



(a) Class I MHC 




** MHC class I and class 
tides, (a) Space-filling model of hurr 
(white) with peptide (red) from HIV 
s 309-31/) 



i in blue. Residues above the peptide are from the i 



with bound pep- 
s HLA-A2 

-oglobulin is 



those below from ct2. (b) Space-filling model of human class II mol- 
ecules HLA-DR1 with the DRa chain shown in white and the DRp 
chain in blue. The peptide (red) in the binding groove is from in- 
fluenza hemagglutinin (amino acid residues 306-318). [From D. A. 



Vignali and J. Strominger, 1994, The 



logist 2:7J2J 



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8536d:Goldsby et al . / ] 



In a critical study of peptide binding by MHC molecules, 
peptides bound by two allelic variants of a class I MHC mol- 
ecule were released chemically and analyzed by HPLC mass 
spectrometry. More than 2000 distinct peptides were found 
among the peptide ligands released from these two class I 
MHC molecules. Since there are approximately 10 5 copies of 
each class I allelic variant per cell, it is estimated that each of 
the 2000 distinct peptides is presented with a frequency of 
100-4000 copies per cell. Evidence suggests that as few as 
100 peptide-MHC complexes are sufficient to target a cell 
for recognition and lysis by a cytotoxic T lymphocyte with a 
receptor specific for this target structure. 

The bound peptides isolated from different class I mole- 
cules have been found to have two distinguishing features: 
they are eight to ten amino acids in length, most commonly 
nine, and they contain specific amino acid residues that ap- 
pear to be essential for binding to a particular MHC mole- 
cule. Binding studies have shown that nonameric peptides 
bind to class I molecules with a 100- to 1000-fold higher 
affinity than do peptides that are either longer or shorter, 
suggesting that this peptide length is most compatible with 
the closed-ended peptide-binding cleft in class I molecules. 
The ability of an individual class I MHC molecule to bind to 
a diverse spectrum of peptides is due to the presence of the 
same or similar amino acid residues at several defined posi- 
tions along the peptides (Figure 7-11). Because these amino 
acid residues anchor the peptide into the groove of the 
MHC molecule, they are called anchor residues. The side 
chains of the anchor residues in the peptide are comple- 
mentary with surface features of the binding cleft of the 
class I MHC molecule. The amino acid residues lining the 
binding sites vary among different class I allelic variants and 



H3N<y><^<P><Q><K><NKiXN><^COO- 

H,N <S><GK5><^<KXAXiXAXL>COO- 
H 3 N ^V><G><pXsXgKkKyK?XT>COO- 
H 3 N^KGKp><i>^XlXiXEXi>COO- 

H 3 N<i>^<iXpXiXiXiK5>#-coo- 

- H 3 N <T>^KQKR><TX^<A><E>#-COO- 
H3N^X^}{^Xi}{N}{NXi>COO- 



peptides eluted from two class I MHC molecules. Anchor residues 
that interact with the class I MHC molecule tend to be hydrophobic 
amino acids. [Data from V. H. Engelhard, 1994, Curr. Opin. Immunol. 
6:13.] 



determine the identity of the anchor residues that can inter- 
act with the molecule. 

All peptides examined to date that bind to class I mole- 
cules contain a carboxyl-terminal anchor. These anchors are 
generally hydrophobic residues (e.g., leucine, isoleucine), al- 
though a few charged amino acids have been reported. Be- 
sides the anchor residue found at the carboxyl terminus, 
another anchor is often found at the second or second and 
third positions at the amino-terminal end of the peptide (see 
Figure 7-11). In general, any peptide of correct length that 
contains the same or similar anchor residues will bind to the 
same class I MHC molecule. The discovery of conserved an- 
chor residues in peptides that bind to various class I MHC 
molecules may permit prediction of which peptides in a 
complex antigen will bind to a particular MHC molecule, 
based on the presence or absence of these motifs. 

X-ray crystallographic analyses of peptide-class I MHC 
complexes have revealed how the peptide-binding cleft in a 
given MHC molecule can interact stably with a broad spec- 
trum of different peptides. The anchor residues at both ends 
of the peptide are buried within the binding cleft, thereby 
holding the peptide firmly in place (Figure 7- 12) . As noted al- 
ready, nonameric peptides are bound preferentially; the main 
contacts between class I MHC molecules and peptides in- 
volve residue 2 at the amino-terminal end and residue 9 at the 
carboxyl terminus of the nonameric peptide. Between the an- 
chors the peptide arches away from the floor of the cleft in the 
middle (Figure 7-13), allowing peptides that are slightly 
longer or shorter to be accommodated. Amino acids that arch 
away from the MHC molecule are more exposed and pre- 
sumably can interact more directly with the T-cell receptor. 

CLASS II MHC-PEPTIDE INTERACTION 
Class II MHC molecules bind peptides and present these 
peptides to CD4 + T cells. Like class I molecules, molecules of 
class II can bind a variety of peptides. In general, these pep- 
tides are derived from exogenous proteins (either self or 
nonself), which are degraded within the endocytic process- 
ing pathway (see Chapter 8). Most of the peptides associated 
with class II MHC molecules are derived from membrane- 
bound proteins or proteins associated with the vesicles of the 
endocytic processing pathway. The membrane-bound pro- 
teins presumably are internalized by phagocytosis or by 
receptor-mediated endocytosis and enter the endocytic pro- 
cessing pathway at this point. For instance, peptides derived 
from digestion of membrane-bound class I MHC molecules 
often are bound to class II MHC molecules. 

Peptides recovered from class II MHC-peptide com- 
plexes generally contain 13-18 amino acid residues, some- 
what longer than the nonameric peptides that most 
commonly bind to class I molecules. The peptide-binding 
cleft in class II molecules is open at both ends (see Figure 
7-10b), allowing longer peptides to extend beyond the ends, 
like a long hot dog in a bun. Peptides bound to class II MHC 
molecules maintain a roughly constant elevation on the 



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Generation of B-Cell and T-Cell Response 




Model of the 

depicting the complex formed with a vesicular stomatitis virus (VSV- 
8) peptide (left, yellow backbone) and Sendai virus (SEV-9) nucleo- 
protein (right, blue backbone). Water molecules (blue spheres) 
interact with the bound peptides. The majority of the surface of both 
peptides is inaccessible for direct contact with T cells (VSV-8 is 83% 
buried; SEV-9 is 75% buried). The H-2K 1 ' surface in the two com- 
plexes exhibits a small, but potentially significant, conformational 
variation, especially in the central region of the binding cleft on the 
right side of the peptides, which corresponds to the a helix in the a2 
domain (see Figure 7-6b). [From M. Matsumura et al., 1992, Science 
257:927; photographs courtesy of D. H. Fremont, M. Matsumura, 
M. Pique, and I. A. Watson.} 



floor of the binding cleft, another feature that distinguishes 
peptide binding to class I and class II molecules. 

Peptide binding studies and structural data for class II 
molecules indicate that a central core of 13 amino acids deter- 
mines the ability of a peptide to bind class II. Longer peptides 
may be accommodated within the class II cleft, but the bind- 
ing characteristics are determined by the central 13 residues. 
The peptides that bind to a particular class II molecule often 
have internal conserved "motifs," but unlike class I-binding 
peptides, they lack conserved anchor residues. Instead, hydro- 
gen bonds between the backbone of the peptide and the class 
II molecule are distributed throughout the binding site rather 
than being clustered predominantly at the ends of the site as 
for class I-bound peptides. Peptides that bind to class II MHC 
molecules contain an internal sequence comprising 7-10 
amino acids that provide the major contact points. Generally, 
this sequence has an aromatic or hydrophobic residue at the 
amino terminus and three additional hydrophobic residues in 
the middle portion and carboxyl-terminal end of the peptide. 






In addition, over 30% of the peptides eluted from class II mol- 
ecules contain a proline residue at position 2 and another 
cluster of prolines at the carboxyl-terminal end. 

Class I and Class II Molecules Exhibit 
Diversity Within a Species and Multiple 
Forms Occur in an Individual 



s diversity is exhibited by the MHC molecules 
; and within individuals. This variability 
echoes the diversity of antibodies and T-cell receptors, but 
the source of diversity for MHC molecules is not the same. 
Antibodies and T-cell receptors are generated by several so- 
matic processes, including gene rearrangement and somatic 
mutation of rearranged genes (see Table 5-2). Thus, the gen- 
eration of T and B cell receptors is dynamic, changing over 
time within an individual. By contrast, the MHC molecules 
expressed by an individual are fixed in the genes and do not 
change over time. The diversity of the MHC within a species 
stems from polymorphism, the presence of multiple alleles at 
a given genetic locus within the species. Diversity of MHC 
molecules in an individual results not only from having dif- 
ferent alleles of each gene but also from the presence of du- 
plicated genes with similar or overlapping functions, not 
unlike the isotypes of immunoglobulins. Because it includes 
genes with similar, but not identical structure and function 
(for example, HLA-A, -B, and -C), the MHC may be said to 
be polygenic. 

The MHC possesses an extraordin 
different alleles at each locus and is c 
morphic genetic complexes known i 
These alleles differ in their DNA sequ 
vidual to another by 5% to 10%. The number of amino acid 
differences between MHC alleles can be quite significant, 
with up to 20 amino acid residues contributing to the 
unique structural nature of each allele. Analysis of human 
HLA class I genes has revealed, as of early 2002, approxi- 
mately 240 A alleles, 470 B alleles, and 1 10 C alleles. In mice, 
the polymorphism is similarly enormous. The human class 
II genes are also highly polymorphic and, in some cases, 
there are different gene numbers in different individuals. 
The number of HLA-DR beta-chain genes may vary from 2 
to 9 in different haplotypes, and approximately 350 alleles of 
DRB genes have been reported. Interestingly, the DRA chain 
is highly conserved, with only 2 different alleles reported. 
Current estimates of actual polymorphism in the human 
MHC are probably on the low side because the most detailed 
data were obtained from populations of European descent. 
The fact that many non-European population groups can- 
not be typed using the MHC serologic typing reagents avail- 
able indicates that the worldwide diversity of the MHC 
genes is far greater. Now that MHC genes can be sequenced 
directly, it is expected that many additional alleles will be 
detected. 

This enormous polymorphism results in a tremendous 
diversity of MHC molecules within a species. Using the num- 
bers given above for the allelic forms of human HLA-A, -B, 



rily large number of 
ne of the most poly- 
l higher vertebrates. 
:nces from one indi- 



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Major Histocompatibility Complex chapter 7 173 





' Conformati 
molecules, (a) Schematic c 
bound peptides of different lengths. Longer 
middle, whereas shorter peptides are more i 
the M HC molecule is by hydrogen bonds to a 
8/9. (b) Molecular models based on crystal st 
virus antigenic peptide (blue) and an endoge 
bound to a class I MHC molecule. Residues 
numbers corresponding to those in part (a). (( 
and «2 domains of HLA-B27 and a bound a 
on x-ray crystallographic analysis of the cocrystallized peptide-HLA 
molecule. The peptide (purple) arches up away from the B strands 
forming the floor of the binding cleft and interacts with twelve water 
molecules (spheres). [Part (a) adapted from P. Parham, 1992, Nature 
360:300, © 1992 Macmillan Magazines Limited; part (b) adapted 
from M. L. Silver et a!., 1992, Nature 360:367, © 1992 Macmillan 
Magazines Limited; part (c) adapted from D. R. Madden et al., 1992, 
Cell 70:1035, reprinted by permission of Cell Press.] 



of peptides bound to class I MHC 
ram of conformational difference in 
peptides bulge in the 
xtended. Contact with 
ichor residues 1/2 and 
'ucture of an influenza 
nous peptide (purple) 
are identified by small 
) Representation of al 
itigenic peptide based 



and -C, we can calculate the theoretical number of combina- 
tions that can exist by multiplying 240 X 470 X 110, yielding 
upwards of 12 million different class I haplotypes possible in 
the population. If class II loci are considered, the 5 DRB 
genes Bl through B5 have 304, 1, 35, 11, and 15 alleles re- 
spectively, DQA1 and Bl contribute 22 and 49 alleles, respec- 
tively and, DPB1 96 alleles; this allows approximately 1.8 X 
10 11 different class II combinations. Because each haplotype 
contains both class I and class II genes, the numbers are mul- 
tiplied to give a total of 2.25 X 10 18 possible combinations of 
these class I and II alleles. 

LINKAGE DISEQUILIBRIUM 

The calculation of theoretical diversity in the previous para- 
graph assumes completely random combinations of alleles. 
The actual diversity is known to be less, because certain allelic 
combinations occur more frequently in HLA haplotypes 
than predicted by random combination, a state referred to as 
linkage disequilibrium. Briefly, linkage disequilibrium is the 
difference between the frequency observed for a particular 
combination of alleles and that expected from the frequencies 
of the individual alleles. The expected frequency for the com- 
bination may be calculated by multiplying the frequencies of 



the two alleles. For example, if HLA-A1 occurs in 16% of in- 
dividuals in a population (frequency = 0.16) and HLA-B8 in 
9% of that group (frequency = 0.09) it is expected that about 
1.4% of the group should have both alleles (0.16 X 0.09 = 
0.014). However, the data show that HLA-A1 and HLA-B8 
are found together in 8.8% of individuals studied. This dif- 
ference is a measure of the linkage disequilibrium between 
these alleles of class I MHC genes. 

Several explanations have been advanced to explain link- 
age disequilibrium. The simplest is that too few generations 
have elapsed to allow the number of crossovers necessary to 
reach equilibrium among the alleles present in founders of 
the population. The haplotypes that are over-represented in 
the population today would then reflect the combinations of 
alleles present in the founders. Alternatively, selective effects 
could also result in the higher frequency of certain allelic 
combinations. For example, certain combinations of alleles 
might produce resistance to certain diseases, causing them to 
be selected for and over-represented, or they might generate 
harmful effects, such as susceptibility to autoimmune disor- 
ders, and undergo negative selection. A third hypothesis is 
that crossovers are more frequent in certain DNA sequence 
regions, and the presence or absence of regions prone 
to crossover (hotspots) between alleles can dictate the 



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Generation of B-Cell and T-Cell Respor 



frequency of allelic association. Data in support of this was 
found in mouse breeding studies that generated new recom- 
binant H-2 types. The points of crossover in the new MHC 
haplotypes were not randomly distributed throughout the 
complex. Instead, the same regions of crossover were found 
in more than one recombinant haplotype. This suggests that 
hotspots of recombination do exist that would influence 
linkage disequilibrium in populations. 

Despite linkage disequilibrium, there is still enormous poly- 
morphism in the human MHC, and it remains very difficult to 
match donor and acceptor MHC types for successful organ 
transplants. The consequences of this major obstacle to the 
therapeutic use of transplantation are described in Chapter 21. 

FUNCTIONAL RELEVANCE OF MHC POLYMORPHISM 
Sequence divergence among alleles of the MHC within a 
species is very high, as great as the divergence observed for 
the genes encoding some enzymes across species. Also of in- 
terest is that the sequence variation among MHC molecules 
is not randomly distributed along the entire polypeptide 
chain but instead is clustered in short stretches, largely 
within the membrane-distal al and a2 domains of class I 



molecules (Figure 7-14a). Similar patterns of diversity are 
observed in the al and (52 domains of class II molecules. 

Progress has been made in locating the polymorphic 
residues within the three-dimensional structure of the mem- 
brane-distal domains in class I and class II MHC molecules 
and in relating allelic differences to functional differences 
(Figure 7- 14b). For example, of 17 amino acids previously 
shown to display significant polymorphism in the HLA-A2 
molecule, 1 5 were shown by x-ray crystallographic analysis to 
be in the peptide-binding cleft of this molecule. The location 
of so many polymorphic amino acids within the binding site 
for processed antigen strongly suggests that allelic differences 
contribute to the observed differences in the ability of MHC 
molecules to interact with a given antigenic peptide. 



Detailed Genomic Map 
of MHC Genes 

The MHC spans some 2000 kb of mouse DNA and some 
4000 kb of human DNA. The recently completed human 
genome sequence shows this region to be densely packed 




20 40 60 



100 120 140 160 180 200 220 240 260 
Residue number 




|Q (a) Plots of variability in the ar 


nino acid sequence of 


c class 1 MHC molecules in humans vers 


s residue position. In 


xternal domains, most of the variable res 


dues are in the mem- 


e-distal al and a.2 domains, (b) Location 


}f polymorphic amino 


residues (red) in the a1/ot2 domain of a 


human class 1 MHC 


cule. [Part (a) adapted from R. Sodoyer 


et al., 1984, EM BO j. 



3:879, reprinted by permission of Oxford University Press; part 
(b) adapted, with permission, from P. Parham, 1989, Nature 342:617, 
© 1989 Macmillan Magazines Limited.] 



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Major Histocompatibility Complex chapter 7 175 



MOUSE CHROMOSOME 17 
Complex < 



50kb 



-H 1 >< 1 ><- 



HUMAN CHROMOSOME 6 



■■0-DtHHh-O-S 



Centromere 









C2, C4A, C4B, Bf 
CYP21,CYP21P 

Gla/b 
HSP 

LMP2, LMP7 
TAP1, TAP2 
TNP-a, TNF-fi 



Encoded protein 
Complement components 
Steroid 21-hydroxylases 
Valyl-tRNA synthetase 
Heat-shock protein 
Proteasomc-lik 



eluding genes encoding classical and noncl; 
I MHC genes are colored red, MHC II gem 
MHC III are colored green. Classical class I | 
blue, and the nonclassical MHC genes are 
classical and nonclassical does not apply to 
proteins encoded by the nonclassical class 
there are nonclassical genes located di 



*Now designated HFE 

le mouse and human MHC, in- 
.sical MHC molecules. The class 
; are colored blue, and genes in 
?nes are labeled in red, class II in 
abeled in black. The concept of 
lass III. The functions for certain 
genes are known. In the mouse, 



with genes, most of which have known functions. Our cur- 
rent understanding of the genomic organization of mouse 
and human MHC genes is diagrammed in Figure 7-15. 

The Human Class I Region Spans 
about 2000 kb at the Telomeric End 
of the HLA Complex 

In humans, the class I MHC region is about 2000 kb long 
and contains approximately 20 genes. In mice, the class I 
MHC consists of two regions separated by the intervening 
class II and class III regions. Included within the class I re- 
gion are the genes encoding the well-characterized classical 
class I MHC molecules designated HLA-A, HLA-B, and 
HLA-C in humans and H-2K, H-2D, and H-2L in mice. 
Many nonclassical class I genes, identified by molecular 



mapping, also are present in both the mouse and human 
MHC. In mice, the nonclassical class I genes are located in 
three regions (H-2Q, T, and M) downstream from the H-2 
complex (M is not shown in Figure 7-15). In humans, the 
nonclassical class I genes include the HLA-E, HLA-F, HLA-G, 
HFE, HLA-J, and HLA -X loci as well as a recently discovered 
family of genes called MIC, which includes MICA through 
MICE. Some of the nonclassical class I MHC genes are 
pseudogenes and do not encode a protein product, but oth- 
ers, such as HLA-G and HFE, encode class I— like products 
with highly specialized functions. The MIC family of class I 
genes has only 15%-30% sequence identity to classical class 
I, and those designated as MICA are highly polymorphic. 
The MIC gene products are expressed at low levels in epithe- 
lial cells and are induced by heat or other stimuli that influ- 
ence heat shock proteins. 



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Generation of B-Cell and T-Cell Respor 



The functions of the nonclassical class I MHC molecules 
remain largely unknown, although a few studies suggest that 
some of these molecules, like the classical class I MHC mol- 
ecules, may present peptides to T cells. One intriguing find- 
ing is that the mouse molecule encoded by the H-2Mlocus is 
able to bind a self-peptide derived from a subunit of NADH 
dehydrogenase, an enzyme encoded by the mitochondrial 
genome. This particular self-peptide contains an amino- 
terminal formylated methionine. What is interesting about 
this finding is that peptides derived from prokaryotic organ- 
isms often have formylated amino -terminal methionine 
residues. This H-2M-encoded class I molecule may thus be 
uniquely suited to present peptides from prokaryotic organ- 
isms that are able to grow intracellularly. Such organisms in- 
clude Mycobacterium tuberculosis, Listeria monocytogenes, 
Brucella abortus, and Salmonella typhimurium. 

Up to this point, all description of antigen presentation 
by class I and class II molecules has been confined to presen- 
tation of peptide antigens. As will be seen in the description 
of antigen presentation (Chapter 8), there are also molecules 
with structural similarity to class I molecules that present 
non-peptide antigens, such as glycolipids, to T cells. A major 
family of such molecules, designated CD1, has been shown 
to present lipid antigens derived from bacteria. The CD1 
molecules are not encoded within the MHC but are located 
on chromosome 1. 

The Class II MHC Genes Are Located 
at the Centromeric End of H LA 

The class II MHC region contains the genes encoding the a 
and p chains of the classical class II MHC molecules desig- 
nated HLA-DR, DP, and DQ in humans and H-2IA and -IE 
in mice. Molecular mapping of the class II MHC has re- 
vealed multiple (3-chain genes in some regions in both mice 
and humans, as well as multiple a-chain genes in humans 
(see Figure 7-15). In the human DR region, for example, 
there are three or four functional p-chain genes. All of the p- 
chain gene products can be expressed together with the a- 
chain gene product in a given cell, thereby increasing the 
number of different antigen-presenting molecules on the 
cell. Although the human DR region contains just one a- 
chain gene, the DP and DQ regions each contains two. 

Genes encoding nonclassical class II MHC molecules 
have also been identified in both humans and mice. In mice, 
several class II genes (Oa, 0|3, Ma, and M(3) encode non- 
classical MHC molecules that exhibit limited polymorphism 
and a different pattern of expression than the classical IA 
and IE class II molecules. In the human class II region, non- 
classical genes designated DM and DO have been identified. 
The DM genes encode a class II— like molecule (HLA-DM) 
that facilitates the loading of antigenic peptides into the class 
II MHC molecules. Class II DO molecules, which are ex- 
pressed only in the thymus and mature B cells, have been 
shown to serve as regulators of class II antigen processing. 
The functions of HLA-DM and HLA-DO will be described 
further in Chapter 8. 



Human MHC Class III Genes 
Are Between Class I and II 

The class III region of the MHC in humans and mice con- 
tains a heterogeneous collection of genes (see Figure 7-15). 
These genes encode several complement components, two 
steroid 21 -hydroxylases, two heat-shock proteins, and two 
cytokines (TNF-a and TNF-0). Some of these class III MHC 
gene products play a role in certain diseases. For example, 
mutations in the genes encoding 21 -hydroxylase have been 
linked to congenital adrenal hyperplasia. Interestingly, the 
presence of a linked class III gene cluster is conserved in all 
species with an MHC region. 



Cellular Distribution 
of MHC Molecules 

In general, the classical class I MHC molecules are expressed 
on most nucleated cells, but the level of expression differs 
among different cell types. The highest levels of class I mole- 
cules are expressed by lymphocytes, where they constitute 
approximately 1% of the total plasma-membrane proteins, 
or some 5 X 10 5 molecules per cell. In contrast, fibroblasts, 
muscle cells, liver hepatocytes, and neural cells express very 
low levels of class I MHC molecules. The low level on liver 
cells may contribute to the considerable success of liver 
transplants by reducing the likelihood of graft recognition 
by T c of the recipient. A few cell types (e.g., neurons and 
sperm cells at certain stages of differentiation) appear to lack 
class I MHC molecules altogether. 

As noted earlier, any particular MHC molecule can bind 
many different peptides. Since the MHC alleles are codomi- 
nantly expressed, a heterozygous individual expresses on its 
cells the gene products encoded by both alleles at each MHC 
locus. An F : mouse, for example, expresses the K, D, and L 
from each parent (six different class I MHC molecules) on 
each of its nucleated cells (Figure 7-16). A similar situation 
occurs in humans; that is, a heterozygous individual ex- 
presses the A, B, and C alleles from each parent (six different 
class I MHC molecules) on the membrane of each nucleated 
cell. The expression of so many class I MHC molecules al- 
lows each cell to display a large number of peptides in the 
peptide-binding clefts of its MHC molecules. 

In normal, healthy cells, the class I molecules will display 
self-peptides resulting from normal turnover of self pro- 
teins. In cells infected by a virus, viral peptides, as well as self- 
peptides, will be displayed. A single virus-infected cell 
should be envisioned as having various class I molecules on 
its membrane, each displaying different sets of viral pep- 
tides. Because of individual allelic differences in the peptide- 
binding clefts of the class I MHC molecules, different 
individuals within a species will have the ability to bind dif- 
ferent sets of viral peptides. 

Unlike class I MHC molecules, class II molecules are ex- 
pressed constitutively only by antigen-presenting cells, pri- 



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Major Histocompatibility Complex 




Class II IE« rf p fe IAa"P" 

molecules 

pressed on antigen-presenting cells of a heterozygous \-\-2 k/d mouse. 
Both the maternal and paternal MHC genes are expressed. Because 
the class II molecules are heterodimers, heterologous molecules 
containing one maternal-derived and one paternal-derived chain are 
produced. The B 2 -microglobulin component of class I molecules 
(pink) is encoded by a gene on a separate chromosome and may be 
derived from either parent. 



marily macrophages, dendritic cells, and B cells; thymic 
epithelial cells and some other cell types can be induced to 
express class II molecules and to function as antigen-pre- 
senting cells under certain conditions and under stimulation 
of some cytokines (see Chapter 8). Among the various cell 
types that express class II MHC molecules, marked differ- 
ences in expression have been observed. In some cases, class 
II expression depends on the cell's differentiation stage. For 
example, class II molecules cannot be detected on pre-B cells 
but are expressed constitutively on the membrane of mature 
B cells. Similarly, monocytes and macrophages express only 
low levels of class II molecules until they are activated by in- 
teraction with an antigen, after which the level of expression 
increases significantly. 

Because each of the classical class II MHC molecules is 
composed of two different polypeptide chains, which are en- 
coded by different loci, a heterozygous individual expresses 
not only the parental class II molecules but also molecules 
containing a and p chains from different chromosomes. For 
example, an H-2 mouse expresses IA and IE class II mole- 
cules; similarly, an H-2 mouse expresses IA and IE mole- 
cules. The Fi progeny resulting from crosses of mice with 
these two haplotypes express four parental class II molecules 
and four molecules containing one parent's a chain and the 
other parent's p chain (as shown in Figure 7-16). Since the 
human MHC contains three classical class II genes {DP, DQ, 
and DR), a heterozygous individual expresses six parental 
class II molecules and six molecules containing a and (3 chain 



combinations from either parent. The number of different 
class II molecules expressed by an individual is increased fur- 
ther by the presence of multiple p-chain genes in mice and 
humans, and in humans by multiple a-chain genes. The di- 
versity generated by these mechanisms presumably increases 
the number of different antigenic peptides that can be pre- 
sented and thus is advantageous to the organism. 



Regulation of MHC Expression 

Research on the regulatory mechanisms that control the dif- 
ferential expression of MHC genes in different cell types is 
still in its infancy, but much has been learned. The publica- 
tion of the complete genomic map of the MHC complex is 
expected to greatly accelerate the identification and investi- 
gation of coding and regulatory sequences, leading to new 
directions in research on how the system is controlled. 

Both class I and class II MHC genes are flanked by 5' pro- 
moter sequences, which bind sequence-specific transcrip- 
tion factors. The promoter motifs and transcription factors 
that bind to these motifs have been identified for a number 
of MHC genes. Transcriptional regulation of the MHC is 
mediated by both positive and negative elements. For exam- 
ple, an MHC II transactivator, called CIITA, and another 
transcription factor, called RFX, both have been shown to 
bind to the promoter region of class II MHC genes. Defects 
in these transcription factors cause one form of bare lym- 
phocyte syndrome (see the Clinical Focus box in Chapter 8). 
Patients with this disorder lack class II MHC molecules on 
their cells and as a result suffer a severe immunodeficiency 
due to the central role of class II MHC molecules in T-cell 



The expression of MHC molecules is also regulated by 
various cytokines. The interferons (alpha, beta, and gamma) 
and tumor necrosis factor have each been shown to increase 
expression of class I MHC molecules on cells. Interferon 
gamma (IFN-7), for example, appears to induce the forma- 
tion of a specific transcription factor that binds to the pro- 
moter sequence flanking the class I MHC genes. Binding of 
this transcription factor to the promoter sequence appears 
to coordinate the up-regulation of transcription of the genes 
encoding the class I a chain, p 2 -microglobulin, the protea- 
some subunits (LMP), and the transporter subunits (TAP). 
IFN-~y also has been shown to induce expression of the class 
II transactivator (CIITA), thereby indirectly increasing ex- 
pression of class II MHC molecules on a variety of cells, in- 
cluding non-antigen-presenting cells (e.g., skin keratin- 
ocytes, intestinal epithelial cells, vascular endothelium, pla- 
cental cells, and pancreatic beta cells). Other cytokines influ- 
ence MHC expression only in certain cell types; for example, 
IL-4 increases expression of class II molecules by resting B 
cells. Expression of class II molecules by B cells is down-reg- 
ulated by IFN-7; corticosteroids and prostaglandins also de- 
crease expression of class II molecules. 

MHC expression is decreased by infection with certain 
viruses, including human cytomegalovirus (CMV), hepatitis 



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Generation of B-Cell and T-Cell Respor 



B virus (HBV), and adenovirus 12 (Adl2). In some cases, re- 
duced expression of class I MHC molecules on cell surfaces 
is due to decreased levels of a component needed for peptide 
transport or MHC class I assembly rather than in transcrip- 
tion. In cytomegalovirus infection, for example, a viral pro- 
tein binds to p 2 - m i cro gl°bulin, preventing assembly of class 
I MHC molecules and their transport to the plasma mem- 
brane. Adenovirus 12 infection causes a pronounced de- 
crease in transcription of the transporter genes (TAP1 and 
TAP2). As the next chapter describes, the TAP gene products 
play an important role in peptide transport from the cyto- 
plasm into the rough endoplasmic reticulum. Blocking of 
TAP gene expression inhibits peptide transport; as a result, 
class I MHC molecules cannot assemble with (^-microglob- 
ulin or be transported to the cell membrane. Decreased ex- 
pression of class I MHC molecules, by whatever mechanism, 
is likely to help viruses evade the immune response by re- 
ducing the likelihood that virus-infected cells can display 
MHC-viral peptide complexes and become targets for CTL- 
mediated destruction. 



MHC and Immune Responsiveness 

Early studies by B. Benacerraf in which guinea pigs were im 
munized with simple synthetic antigens were the first tc 
show that the ability of an animal to mounl 



recombin 



nated Ir c 



sponse, as measured by the production of serum antibodies, 
is determined by its MHC haplotype. Later experiments by 
H. McDevitt, M. Sela, and their colleagues used congenic and 
nt congenic mouse strains to map the control of 
sponsiveness to class II MHC genes. In early re- 
^enes responsible for this phenotype were desig- 
r immune response genes, and for this reason 
mouse class II products are called IA and IE. We now know 
that the dependence of immune responsiveness on the class 
II MHC reflects the central role of class II MHC molecules in 
presenting antigen to T H cells. 

Two explanations have been proposed to account for the 
variability in immune responsiveness observed among dif- 
ferent haplotypes. According to the determinant-selection 
model, different class II MHC molecules differ in their abil- 
ity to bind processed antigen. According to the alternative 
holes-in-the-repertoire model, T cells bearing receptors that 
recognize foreign antigens closely resembling self-antigens 
may be eliminated during thymic processing. Since the T- 
cell response to an antigen involves a trimolecular complex 
of the T cell's receptor, an antigenic peptide, and an MHC 
molecule (see Figure 3-8), both models may be correct. 
That is, the absence of an MHC molecule that can bind and 
present a given peptide, or the absence of T-cell receptors 
that can recognize a given peptide-MHC molecule com- 
plex, could result in the absence of immune responsiveness 
for the observed relationship between 



PERCENTAGE OF LABELED PEPTIDE BOUND TO* 



Labeled peptide* 

Ovalbumin (323-339) 
Influenza hemagglutinin (130-142) 
Hen egg-white lysozyme (46-61) 
Hen egg-white lysozyme (74-86) 
Hen egg-white lysozyme (81-96) 
Myoglobin (132-153) 
Pigeon cytochrome c (88-104) 
\ repressor (12-26)* 



MHC restriction 
ofresponders 1 



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Major Histocompatibility Complex 



MHC haplotype and immune responsiveness to exogenous 
antigens. 

According to the determinant-selection model, the MHC 
polymorphism within a species will generate a diversity of 
binding specificities, and thus different patterns of respon- 
siveness to antigens. If this model is correct, then class II 
MHC molecules from mouse strains that respond to a par- 
ticular antigen and those that do not should show differen- 
tial binding of that antigen. Table 7-3 presents data on the 
binding of various radiolabeled peptides to class II IA and IE 
molecules with the H-2 d or \l-2 k haplotype. Each of the 
listed peptides binds significantly to only one of the IA or IE 
molecules. Furthermore, in all but one case, the haplotype of 
the class II molecule showing the highest affinity for a par- 
ticular peptide is the same as the haplotype of responder 
strains for that peptide, as the determinant-selection model 
predicts. 

The single exception to the general pattern in Table 7-3 
(residues 12-26 of the X repressor protein) gives evidence 
that the influence on immune responsiveness can also be 
caused by absence of functional T cells (holes-in-the-reper- 
toire model) capable of recognizing a given antigen-MHC 
molecule complex. The \ repressor peptide binds best in 
vitro to IE d , yet the MHC restriction for response to this pep- 



tide is known to be associated not with IE' but instead witl 
LA and IE . This suggests that T cells recognizing this re 
pressor peptide in association with IE may have been elim 
inated by negative selection in the thymus, leaving a hole ii 
the T-cell repertoire. 



MHC and Disease Susceptibility 

Some HLA alleles occur at a much higher frequency in those 
suffering from certain diseases than in the general popula- 
tion. The diseases associated with particular MHC alleles 
include autoimmune disorders, certain viral diseases, 
disorders of the complement system, some neurologic disor- 
ders, and several different allergies. The association between 
HLA alleles and a given disease may be quantified by deter- 
mining the frequency of the HLA alleles expressed by indi- 
viduals afflicted with the disease, then comparing these data 
with the frequency of the same alleles in the general popula- 
tion. Such a comparison allows calculation of relative risk 
(see Table 7-4). A relative risk value of 1 means that the HLA 
allele is expressed with the same frequency in the patient and 
general populations, indicating that the allele confers no in- 
creased risk for the disease. A relative risk value substantially 






H 



Associated HLA allele 



Ankylosing spondylitis 
Goodpasture's syndrome 
Gluten-sensitive enteropathy 
Hereditary hemochromatosis 



Insulin-dependent diabetes 
Multiple sclerosis 
Myasthenia gravis 
Narcolepsy 
Reactive arthritis 



Salmonella, Gonococcus) 



Reite 



syndro 



Rheumatoid arthritis 
Sjogren's syndrome 
Systemic lupus eryther 



DR2 
DR3 



B14 



A3/B14 

DR4/DR3 

DR2 

DR3 

DR2 

B27 



ited by dividing the freque 



e, D. C. Dale and D. D. F 



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CLINICAL FOCUS 

HFE and Hereditary 
Hemochromatosis 



Heredita 



hemochro- 
matosis (HH) is a disease in which 
defective regulation of dietary iron ab- 
sorption leads to increased levels of 
iron. HH (which in earlier reports may be 
referred to as idiopathic or primary he- 
known autosomal recessive genetic dis- 
order in North Americans of European 
descent, with a frequency of 3-4 cases 
per 1000 persons. Recent studies show 
that this disease is associated with a mu- 
tation in the nonclassical class I gene 
HFE (formerly designated HLA-H), 
which lies to the telomeric side of HLA-A. 
The association of the HFE gene with 
HH is an example of how potentially life- 
saving clinical information can be ob- 
tained by studying the connection of 
HLA genes with disease. 

The total iron content of a normal hu- 
man adult is 3 to 4 grams; the average di- 
etary intake of iron is about 10 to 20 



milligrams per day; of this, only 1 to 2 mg 
is absorbed. The iron balance is main- 
tained by control of its absorption from di- 
gested food in the intestinal tract. The 
primary defect in HH is increased gas- 
trointestinal uptake of iron and, as a result 
of this, patients with HH may throughout 
their lives accumulate 15 to 35 grams of 



iron instead of the normal 3 to 4 grams. 
The iron overload results in pathologic ac- 
cumulation of iron in cells of many or- 
gans, including the heart and liver. 
Although a severe form of HH may result 
in heart disease in children, the clinical 
manifestations of the disease are not usu- 
ally seen until 40 to 50 years of age. Males 
are affected eight times more frequently 
than females. Early symptoms of HH are 
rather nonspecific and include weakness, 
lethargy, abdominal pain, diabetes, impo- 
tence, and severe joint pain. Physical ex- 
amination of HH sufferers reveals liver 
damage, skin pigmentation, arthritis, en- 




High-magnification iron stain of 
liver cells from HH patient. The 
stain confirms the presence of 
iron in both parenchymal cells 
(thick arrow) and bile duct cells 
(thin arrow). This woman with 
hemochromatosis required 
removal of 72 units (about 36 
liters or 9 gallons) of blood dur- 
ing one and a half years to ren- 
der her liver free of excess iron. 
[SAM CD: A Comprehensive 
Knowledge Base of Internal 
Medicine, D. C. Dale and D. D. 
Federman, eds., 1997, Scientific 
American, New York.] 



above 1 indicates an association between the HLA allele and 
the disease. As Table 7-4 shows, individuals with the HLA- 
B27 allele have a 90 times greater likelihood (relative risk of 
90) of developing the autoimmune disease ankylosing 
spondylitis, an inflammatory disease of vertebral joints 
characterized by destruction of cartilage, than do individu- 
als with a different HLA-B allele. 

The existence of an association between an MHC allele 
and a disease should not be interpreted to imply that the ex- 
pression of the allele has caused the disease — the relationship 
between MHC alleles and development of disease is complex. 
In the case of ankylosing spondylitis, for example, it has been 
suggested that because of the close linkage of the TNF-a and 
TNF-fi genes with the HLA-B locus, these cytokines may be 
involved in the destruction of cartilage. An association of 
HLA class I genes with the disease hereditary hemochro- 
matosis is discussed in the Clinical Focus box in this chapter. 

When the associations between MHC alleles and disease 
are weak, reflected by low relative risk values, it is likely that 
multiple genes influence susceptibility, of which only one is 



in the MHC. That these diseases are not inherited by simple 
Mendelian segregation of MHC alleles can be seen in identi- 
cal twins; both inherit the MHC risk factor, but it is by no 
means certain that both will develop the disease. This find- 
ing suggests that multiple genetic and environmental factors 
have roles in the development of disease, especially autoim- 
mune diseases, with the MHC playing an important but not 
exclusive role. An additional difficulty in associating a par- 
ticular MHC product with disease is the genetic phenome- 
non of linkage disequilibrium, which was described above. 
The fact that some of the class I MHC alleles are in linkage 
disequilibrium with the class II MHC alleles makes their 
contribution to disease susceptibility appear more pro- 
nounced than it actually is. If, for example, DR4 contributes 
to risk of a disease, and if it occurs frequently in combination 
with A3 because of linkage disequilibrium, then A3 would 
incorrectly appear to be associated with the disease. Im- 
proved genomic mapping techniques make it possible to an- 
alyze the linkage between the MHC and various diseases 
more fully and to assess the contributions from other loci. 



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40 AM Page 181 



Major Histocompatibility Complex 



larged spleen, jaundice, and peripheral 
edema. If untreated, HH results in hepatic 
cancer, liver failure, severe diabetes, and 
heart disease. Exactly how the increase in 

not known, but repeated phlebotomy (tak- 
ing blood) is an effective treatment if the 
disease is recognized before there is ex- 
tensive damage to organs. Phlebotomy 
does not reverse damage already done. 
Phlebotomy (also called blood-letting) 
was used as treatment for many condi- 
tions in former times; HH may be one of 
the rare instances in which the treatment 
had a positive rather than a harmful effect 
on the patient. 

Prior to appearance of the recognized 
signs of the disease, such as the charac- 
teristic skin pigmentation or liver dys- 
function, diagnosis is difficult unless for 
some reason (such as family history of 
the disease) HH is suspected and spe- 
cific tests for iron metabolism are per- 
formed. A reliable genetic test for HH 



>uld ; 



nifestatio 



versible organ damage. 

Because it is a common disease, the 
association of HH with HLA was studied; 
initially a significant association with the 
HLA-A3 allele was found (RR of 9.3). This 



association is well documented, but the 
relatively high frequency of the HLA-A3 al- 
lele (present in 20% of the North Ameri- 
can population) makes this an 
inadequate marker; the majority of indi- 
viduals with HLA-A3 will not have HH. 
Further studies showed a greatly in- 
creased relative risk in individuals with 
the combination of HLA-A3 and HLA- 
Bi4; homozygotes for these two alleles 
carried a relative risk for HH of 90. De- 
tailed studies of several populations in 
the US and France with high incidence of 
HH revealed a mutation in the nonclassi- 
cal HLA class I gene HFE in 83%-100% 
of patients with HH. HFE, which lies 
close to the HLA-A locus, was shown in 
several independent studies to carry a 
characteristic mutation at position 283 in 
HH patients, with substitution of a tyro- 
sine residue for the cysteine normally 
found at this position. The substitution 
precludes formation of the disulfide link 
between cysteines in the a3 domain, 
which is necessary for association of the 
MHC a chain with ^-microglobulin and 
for expression on the cell surface. HFE 
molecules are normally expressed on the 
surface of cells in the stomach, in- 
testines, and liver. There is evidence 
showing that HFE plays a role in the abil- 



ity of these organs to regulate iron uptake 
from the circulation. The mechanism by 
which HFE functions involves binding to 
the transferrin receptor, which reduces 
the affinity of the receptor for iron-loaded 
transferrin. This lowers the uptake of iron 
by the cell. Mutations that interfere with 
the ability of HFE to form a complex with 
transferrin and its receptor can lead to in- 
creased iron absorption and HH. 

There are several possible reasons for 
why this defect continues to be so com- 
mon in our population. Factors that favor 
the spread of the defective HFE gene 
would include the fact that it is a reces- 
sive trait, so only homozygotes are af- 
fected; the gene is silent in carriers. In 
addition, even in most homozygotes af- 
fected with HH, the disease does not 
manifest itself until later in life and so 
may have minimal influence on the 
breeding success of the HH sufferer. 

Studies of knockout mice that lack 
the gene for (^-microglobulin demon- 
strate that MHC class I products on cell 
surfaces are necessary for the mainte- 
nance of normal iron metabolism. These 
mice, which are unable to express any of 
their class I molecules on the cell si 
faces, suffer from iron overload with d 
ease consequences similar to HH. 



A number of hypotheses have been offered t< 
the role of the MHC in disease susceptibility. As noted ear- 
lier, allelic differences may yield differences in immune re- 
sponsiveness arising from variation in the ability to present 
processed antigen or the ability of T cells to recognize pre- 
sented antigen. Allelic forms of MHC genes may also encode 
molecules that are recognized as receptors by viruses or bac- 
terial toxins. As will be explained in Chapter 16, the genetic 
analysis of disease must consider the possibility that genes at 
multiple loci may be involved and that complex interactions 
among them may be needed to trigger disease. 

Some evidence suggests that a reduction in MHC poly- 
morphism within a species may predispose that species to 
infectious disease. Cheetahs and certain other wild cats, such 
as Florida panthers, that have been shown to be highly sus- 
ceptible to viral disease have very limited MHC polymor- 
phism. It is postulated that the present cheetah population 
(Figure 7-17) arose from a limited breeding stock, causing a 
loss of MHC diversity. The increased susceptibility of chee- 
tahs to various viral diseases may result from a reduction in 




ah female with two nearly full gro 
Polymorphism in MHC genes of the cheetah is very limited 
ably because of a bottleneck in breeding that occurred in th 
distant past. It is assumed that all cheetahs alive today an 
dants of a very small breeding pool. [Photograph taken in 
vango Delta, Botswana, by T.J. Kindt.] 



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the number of different MHC molecules available to the 
species as a whole and a corresponding limitation on the 
range of processed antigens with which these MHC mole- 
cules can interact. Thus, the high level of MHC polymor- 
phism that has been observed in various species may provide 
the advantage of a broad range of antigen-presenting MHC 
molecules. Although some individuals within a species 
probably will not be able to develop an immune response to 
any given pathogen and therefore will be susceptible to in- 
fection by it, extreme polymorphism ensures that at least 
some members of a species will be able to respond and will 
be resistant. In this way, MHC diversity appears to protect a 
species from a wide range of infectious diseases. 



SUMMARY 

■ The major histocompatibility complex (MHC) comprises 
a stretch of tightly linked genes that encode proteins asso- 
ciated with intercellular recognition and antigen presenta- 
tion to T lymphocytes. 

■ A group of linked MHC genes is generally inherited as a 
unit from parents; these linked groups are called haplo- 
types. 

■ MHC genes are polymorphic in that there are large num- 
bers of alleles for each gene, and they are polygenic in that 
there are a number of different MHC genes. 

■ Class I MHC molecules consist of a large glycoprotein 
chain with 3 extracellular domains and a transmembrane 
segment, and (3 2 -microglobulin, a protein with a single 
domain. 

■ Class II MHC molecules are composed of two noncova- 
lently associated glycoproteins, the a and (3 chain, en- 
coded by separate MHC genes. 

■ X-ray crystallographic analyses reveal peptide-binding 
clefts in the membrane-distal regions of both class I and 
class II MHC molecules. 

■ Both class I and class II MHC molecules present antigen to 
T cells. Class I molecules present processed endogenous 
antigen to CD8 T cells. Class II molecules present pro- 
cessed exogenous antigen to CD4 T cells. 

■ Certain conserved motifs in peptides influence their abil- 
ity to interact with the membrane-distal regions of class I 
and class II MHC molecules. 

■ Class I molecules are expressed on most nucleated cells; 
class II antigens are restricted to B cells, macrophages, and 
dendritic cells. 

■ The class III region of the MHC encodes molecules that 
include a diverse group of proteins that play no role in 
antigen presentation. 

■ Detailed maps of the human and mouse MHC reveal the 
presence of genes involved in antigen processing, includ- 
ing proteasomes and transporters. 

Go to www.whfreeman.com/immunology ^A Self-Test 
Review and quiz of key terms 



■ Studies with mouse strains have shown that MHC haplo- 
type influences immune responsiveness and the ability to 
present antigen. 

■ Increased susceptibility to a number of diseases, predomi- 
nantly, but not exclusively, of an autoimmune nature, has 
been linked to certain MHC alleles. 

References 

Brown, J. H., et al. 1993. Three-dimensional structure of the hu- 
man class II histocompatibility antigen HLA-DR1. Nature 
364:33. 

Drakesmith, H., and A. Townsend. 2000. The 
function of HFE. BioEssays. 22:595. 



■er, A. M., et al. 2001. A genomic v: 
e409:836. 



of immunology. Na- 



International Human Genome Sequencing Coi 
Initial sequencing and analysis of the human ! 



Madden, D. R. 1995. The three-dimensional structure of pep- 
tide-MHC complexes. Annu. Rev. Immunol. 13:587. 

Margulies, D. 1999. The major histocompatibility complex, in 
Fundamental Immunology, 4th ed. W. E. Paul, ed. Lippincott 
Raven, Philadelphia. 

Meyer, D., and G. Thompson. 2001. How selection shapes vari- 
ation of the human major histocompatibility complex: a re- 
view. Ann. Hum. Genet. 65:1. 

Natarajan, K., et al. 1999. MHC class I molecules, structure and 
function. Revs, in Immunogenetics 1:32. 

Parham, P. 1999. Virtual reality in the MHC. Immunol. Revs. 
167:5. 

Rothenberg, B. E., and J. R. Voland. 1996. Beta 2 knockout mice 
develop parenchymal iron overload: A putative role for class I 
genes of the major histocompatibility complex in iron metab- 
olism. Proc. Natl. Acad. Sci. U.S.A. 93:1529. 

Rouas-Freiss, N., et al. 1997. Direct evidence to support the role 
of HLA-G in protecting the fetus from maternal uterine nat- 
ural killer cytolysis. Proc. Natl. Acad. Sci. U.S.A. 94:1 1520. 

Vyse, T. J., and I. A. Todd. 1996. Genetic analysis of ; 
disease. Cell 85:311. 

Yung, Y. C, et al. 2000. The human 
parade of 21 genes at 
day 21:320. 

USEFULWEB SITES 

http://www.bioscience.org/knockout/b2micrgl. 
forbeta-2 microglobulin KO 

http://www.bioscience.org/knockout/mhci.htm 
for MHC class I KO 



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Major Histocompatibility Complex 



http://www.bioscience.org/kn. 
for KO of an MHC class II ch 



http://www.bios 
for KO of the ir 



nce.org/knoi 



This series of destinations in the Bioscience Web site provides 
updated information on studies of the consequences of tar- 
geted disruption of MHC molecules and other component 
molecules including (3 2 microglobulin and the class II invari- 



http://w 



v.bshi.org.uk/ 



British Society for Histocompatibility and Immunogenetics 
home page contains information on tissue typing, transplan- 
tation, and links to worldwide sites concerned with MHC. 

http://www.ebi.ac.uk/imgt/hla/ 

The International ImMunoGeneTics (IMGT) database sec- 
tion contains links concerned with HLA gene structure and 
genetics. It also contains up-to-date listings and sequences for 
all HLA alleles officially recognized by the World Health Or- 
ganization HLA nomenclature ci 



Study Questions 



Clinical Focus Question Almost 90% of Caucasians homozy- 
gous for a mutation in position 283 of the HFE gene have clinical 
signs of hemochromatosis. The fact that 10% of those with the 
mutation are not affected causes a critic of the work to state that 
the HFE is not involved with HH. She contends that this associa- 
tion is just a result of linkage disequilibrium. How would you an- 
swer her? Can you design an experiment to shed further light on 
this association? 

1 . Indicate whether each of the following statements is true or 
false. If you think a statement is false, explain why. 

a. A monoclonal antibody specific for (^-microglobulin 
can be used to detect both class I MHC K and D mole- 
cules on the surface of cells. 

b. Antigen-presenting cells express both class I and class II 
MHC molecules on their membranes. 

c. Class III MHC genes encode membrane-bound proteins. 

d. In outbred populations, an individual is more likely to 
be histocompatible with one of its parents than with its 
siblings. 

e. Class II MHC molecules typically bind to longer peptides 
than do class I molecules. 

f. All cells express class I MHC molecules. 

g. The majority of the peptides displayed by class I and class 
II MHC molecules on cells are derived from self-proteins. 

2. You wish to produce a syngeneic and a congenic mouse 
strain. Indicate whether each of the following characteristics 
applies to production of syngeneic (S), congenic (C), or both 
(S and C) mice. 

a. Requires the greatest number of generations 

b. Requires backcrosses 

c. Yields mice that are genetically identical 

d. Requires selection for homozygosity 



e. Requires sibling crosses 

f. Can be started with outbred mice 

g. Yields progeny that are genetically identical to the parent 
except for a single genetic region 

3. You have generated a congenic A.B mouse strain that has 
been selected for its MHC haplotype. The haplotype of 
strain A was a/a and of strain B was bib. 



. Which s 



l provides the genetic background of this 



b. Which strain provides the haplotype of the MHC of this 
mouse? 

c. To produce this congenic strain, the Fl progeny are al- 
ways backcrossed to which strain? 

d. Why was backcrossing to one of the parents performed? 

e. Why was interbreeding of the F x and F 2 progeny per- 
formed? 

f. Why was selection necessary and what kind of selection 
was performed? 

4. You cross a BALB/c (H-2' ; ) mouse with a CBA (H-2' c ) 
mouse. What MHC molecules will the F[ progeny express on 
(a) its liver cells and (b) its macrophages? 

5. To carry out studies on the structure and function of the 
class I MHC molecule K h and the class II MHC molecule 
IA , you decide to transfect the genes encoding these pro- 
teins into a mouse fibroblast cell line (L cell) derived from 
the C3H strain (H-2 fc ). L cells do not normally function as 
antigen-presenting cells. In the following table, indicate 
which of the listed MHC molecules will ( + ) or will not ( - ) 
be expressed on the membrane of the transfected L cells. 



Transfected gene 


MHC molecules expressed 

on the membrane of the 

transfected L cells 


D k 


D b 


K k 


K» 


IA fc 


\A b 


None 














* 














IAa b 














IA$ b 














IAa h and IA$ h 















6. The SJL mouse strain, which has the H-2 haplotype, has a 
deletion of the IEa locus. 

a. List the classical MHC molecules that are expressed on 
the membrane of macrophages from SJL mice. 

b. If the class II IEa and 7£(3 genes from an H-2 S strain are 
transfected into SJL macrophages, what additional clas- 
sical MHC molecules would be expressed on the trans- 
fected macrophages? 

7. Draw diagrams illustrating the general structure, including 
the domains, of class I MHC molecules, class II MHC mole- 
cules, and membrane-bound antibody on B cells. Label each 



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chain and the domains within it, the antigen-binding regions, 
and regions that have the immunoglobulin-fold structure. 

8. One of the characteristic features of the MHC is the large 
number of different alleles at each locus. 

a. Where are most of the polymorphic amino acid residues 
located in MHC molecules? What is the significance of 
this location? 

b. How is MHC polymorphism thought to be generated? 

9. As a student in an immunology laboratory class, you have 
been given spleen cells from a mouse immunized with the 
LCM virus. You determine the antigen-specific functional ac- 
tivity of these cells with two different assays. In assay 1, the 
spleen cells are incubated with macrophages that have been 
briefly exposed to the LCM virus; the production of inter- 
leukin 2 (IL-2) is a positive response. In assay 2, the spleen 
cells are incubated with LCM-infected target cells; lysis of the 
target cells represents a positive response in this assay. The re- 
sults of the assays using macrophages and target cells of dif- 
ferent haplotypes are presented in the table below. Note that 
the experiment has been set up in a way to exclude alloreac- 
tive responses (reactions against nonself MHC molecules). 

a. The activity of which cell population is detected in each 
of the two assays? 

b. The functional activity of which MHC molecules is de- 
tected in each of the two assays? 

c. From the results of this experiment, which MHC mole- 
cules are required, in addition to the LCM virus, for spe- 
cific reactivity of the spleen cells in each of the two assays? 

d. What additional experiments could you perform to un- 



ambiguously confirm the MHC molecules required for 
antigen-specific reactivity of the spleen cells? 
e. Which of the mouse strains listed in the table below could 
have been the source of the immunized spleen cells tested 
in the functional assays? Give your reasons. 

1 0. A T c -cell clone recognizes a particular measles virus peptide 
when it is presented by H-2D b . Another MHC molecule has 
a peptide-binding cleft identical to the one in H-2D 6 but dif- 
fers from H-2D b at several other amino acids in the al(31 
domain. Predict whether the second MHC molecule could 
present this measles virus peptide to the T c -cell clone. 
Briefly explain your answer. 

1 1 . How can you determine if two different inbred mouse 
strains have identical MHC haplotypes? 

1 2. Human red blood cells are not nucleated and do not express 
any MHC molecules. Why is this property fortuitous for 
blood transfusions? 

13. The hypothetical allelic combination HLA-A99 and HLA- 
B276 carries a relative risk of 200 for a rare, and yet un- 
named, disease that is fatal to pre-adolescent children. 

a. Will every individual with A99/B276 contract the disease? 

b. Will everyone with the disease have the A99/B276 combi- 
nation? 

c. How frequently will the A99/B276 allelic combination be 
observed in the general population? Do you think that 
this combination will be more or less frequent than pre- 
dicted by the frequency of the two individual alleles? 
Why? 



Mouse strain 
macrophages and 










Response of splee 


n cells 


and virus-infected target cells 


IL-2 production in 
response to LCM-pulsed 
macrophages (assay 1) 


Lysis of LCM- 
infected cells 
(assay 2) 


K 


IA IE 


D 


C3H 


k 


k 


k 


k 


+ 


" 


BALB/c 


d 


d 


d 


d 


" 


+ 


(BALB/c X B10.AJF, 


d/k 


d/k 


d/k 


d/d 


+ 


+ 


ATL 


s 


k 


k 


d 


+ 


+ 


B10.A(3R) 


b 


b 


b 


d 


" 


+ 


B10.A(4R) 


k 


k 


~ 


b 


+ 


" 



-A 



49 AM Page 185 ir 



8536d:Goldsby et al . / Immunology 5e-: 



chapter 8 



Antigen Processing 
and Presentation 



rECOGNITION OF FOREIGN PROTEIN ANTIGENS BY 
a T cell requires that peptides derived from the 
antigen be displayed within the cleft of an MHC 
molecule on the membrane of a cell. The formation of these 
peptide-MHC complexes requires that a protein antigen be 
degraded into peptides by a sequence of events called anti- 
gen processing. The degraded peptides then associate with 
MHC molecules within the cell interior, and the peptide- 
MHC complexes are transported to the membrane, where 
they are displayed (antigen presentation). 

Class I and class II MHC molecules associate with pep- 
tides that have been processed in different intracellular com- 
partments. Class I MHC molecules bind peptides derived 
from endogenous antigens that have been processed within 
the cytoplasm of the cell (e.g., normal cellular proteins, tu- 
mor proteins, or viral and bacterial proteins produced 
within infected cells). Class II MHC molecules bind peptides 
derived from exogenous antigens that are internalized by 
phagocytosis or endocytosis and processed within the endo- 
cytic pathway. This chapter examines in more detail the 
mechanism of antigen processing and the means by which 
processed antigen and MHC molecules are combined. In ad- 
dition, a third pathway for the presentation of nonpeptide 
antigens derived from bacterial pathogens is described. 



Self-MHC Restriction of T Cells 

Both CD4 + and CD8 + T cells can recognize antigen only when 
it is presented by a self-MHC molecule, an attribute called self- 
MHC restriction. Beginning in the mid-1970s, experiments 
conducted by a number of researchers demonstrated self- 
MHC restriction in T-cell recognition. A. Rosenthal and E. 
Shevach, for example, showed that antigen-specific prolifera- 
tion of T H cells occurred only in response to antigen presented 
by macrophages of the same MHC haplotype as the T cells. In 
their experimental system, guinea pig macrophages from 
strain 2 were initially incubated with an antigen. After the 
"antigen-pulsed" macrophages had processed the antigen and 
presented it on their surface, they were mixed with T cells from 
the same strain (strain 2), a different strain (strain 13), or 
(2 X 13) ¥ x animals, and the magnitude of T-cell proliferation 
in response to the antigen-pulsed macrophages was measured. 




■ Self-MHC Restriction of T Cells 

■ Role of Antigen-Presenting Cells 

■ Evidence for Two Processing and Presentation 
Pathways 

■ Endogenous Antigens: The Cytosolic Pathway 

■ Exogenous Antigens: The Endocytic Pathway 

■ Presentation of Nonpeptide Antigens 



The results of these experiments, outlined in Figure 8-1, 
showed that strain-2 antigen-pulsed macrophages activated 
strain-2 and ¥ l T cells but not strain- 13 T cells. Similarly, 
strain- 13 antigen-pulsed macrophages activated strain- 13 
and Fj T cells but not strain-2 T cells. Subsequently, congenic 
and recombinant congenic strains of mice, which differed 
from each other only in selected regions of the H-2 complex, 
were used as the source of macrophages and T cells. These ex- 
periments confirmed that the CD4 + T H cell is activated and 
proliferates only in the presence of antigen-pulsed 
macrophages that share class II MHC alleles. Thus, antigen 
recognition by the CD4 + T H cell is class II MHC restricted. 

In 1974 R. Zinkernagel and P. Doherty demonstrated the 
self-MHC restriction of CD8 + T cells. In their experiments, 
mice were immunized with lymphocytic choriomeningitis 
(LCM) virus; several days later, the animals' spleen cells, 
which included T c cells specific for the virus, were isolated 
and incubated with LCM-infected target cells of the same or 
different haplotype (Figure 8-2). They found that the T c cells 
killed only syngeneic virus-infected target cells. Later studies 
with congenic and recombinant congenic strains showed 



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3 AM Page 186 mac79 Mac 79:4 5_BW^f 



Generation of B-Cell and T-Cell Respor 



Strain 2 or 13 
or(2xl3)F 1 



Adherent cells 
Peritoneal macrophages 
Antigen -^ 



VKS^ 



Strain 2 or 13 
or(2xl3)F! 



1 l 7 ^ 

Peritoneal exudate cells Lymph node cells 



T 




H-2* target cells H-2 fe LCM-infected H-2 6 LCM-infected 

target cells target cells 



Antigen-pulsed 
macrophages . 



Measure T-cell 





Antigen-pulsed macrophages 


Tcell 


Strain 2 


Strain 13 


(2 x 13)Fj 


Strain 2 
Strain 13 
(2 x 13)Fj 


; 


\ 


: 



al demonstration of self-MHC 



of 
>r(2 X 13) F, 



itigen-pulsed" 
strain 2, strain 
II proliferation 



T H cells. Peritoneal exudate cells from strain 2, strain 13, 
guinea pigs were incubated in plastic Petri dishes, allowii 
of macrophages, which are adherent cells. The peril 
phages were then incubated with antigen. These "at 
macrophages were incubated in vitro with T cells from s 
13, or (2X13) F, guinea pigs, and the degree of T-c 
was assessed. The results indicated that T H cells could proliferate only 
in response to antigen presented by macrophages that shared MHC al- 
leles. [Adapted pom A. Rosenthal and E. Shevach, 1974, J. Exp. Med. 
138:1194, by copyright permission of the Rockefeller University Press.] 



that the T c cell and the virus-infected target cell must share 
class I molecules encoded by the K or D regions of the MHC. 
Thus, antigen recognition by CD8 + T c cells is class I MHC 



© © © © © 
5 © © 



- 51 Cr release 
(no lysis) 



+ 51 Cr release 
(lysis) 



©© © © © 

-5iCr release 

(no lysis) 



sic experiment of Zinkernagel and Doherty 
demonstrating that antigen recognition by T c cells exhibits MHC re- 
striction. H-2 k mice were primed with the lymphocytic choriomenin- 
gitis (LCM) virus to induce cytotoxic T lymphocytes (CTLs) specific 
for the virus. Spleen cells from this LCM-primed mouse were then 
added to target cells of different H-2 haplotypes that were intracellu- 
lar^ labeled with 5l Cr (black dots) and either infected or not with the 
LCM virus. CTL-mediated killing of the target cells, as measured by 
the release of 51 Cr into the culture supernatant, occurred only if the 
target cells were infected with LCM and had the same MHC haplo- 
type as the CTLs. [Adapted from P. C. Doherty and R. M. Zinkernagel, 
1975, j. Exp. Med. 747:502.] 



restricted. In 1996, Doherty and Zinkernagel were awarded 
the Nobel prize for their major contribution to the under- 
standing of cell-mediated immunity. 



Role of Antigen-Presenting Cells 

As early as 1959, immunologists were confronted with data 
suggesting that T cells and B cells recognized antigen by dif- 
ferent mechanisms. The dogma of the time, which persisted 
until the 1980s, was that cells of the immune system recog- 
nize the entire protein in its native conformation. However, 
experiments by P. G. H. Gell and B. Benacerraf demonstrated 
that, while a primary antibody response and cell-mediated 
response were induced by a protein in its native conforma- 
tion, a secondary antibody response (mediated by B cells) 
could be induced only by native antigen, whereas a secondary 



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:7 9 Mac 79:45_BWrf 



Antigen Processing and Presenta 



cell-mediated response could be induced by either the native 
or the denatured antigen (see Table 3-5). These findings were 
viewed as an interesting enigma, but implications for antigen 
presentation were completely overlooked until the early 
1980s. 

Processing of Antigen Is Required 
for Recognition by T Cells 

The results obtained by K. Ziegler and E. R. Unanue were 
among those that contradicted the prevailing dogma that 
antigen recognition by B and T cells was basically similar. 
These researchers observed that T H -cell activation by bacter- 
ial protein antigens was prevented by treating the antigen- 
presenting cells with paraformaldehyde prior to antigen 
exposure. However, if the antigen-presenting cells were first 
allowed to ingest the antigen and were fixed with paraform- 
aldehyde 1-3 h later, T H -cell activation still occurred (Figure 



8-3a,b). During that interval of 1-3 h, the antigen-presenting 
cells had processed the antigen and had displayed it on the 
membrane in a form able to activate T cells. 

Subsequent experiments by R. P. Shimonkevitz showed 
that internalization and processing could be bypassed if anti- 
gen-presenting cells were exposed to peptide digests of an 
antigen instead of the native antigen (Figure 8-3c). In these 
experiments, antigen-presenting cells were treated with glu- 
taraldehyde (this chemical, like paraformaldehyde, fixes the 
cell, making the membrane impermeable) and then incu- 
bated with native ovalbumin or with ovalbumin that had 
been subjected to partial enzymatic digestion. The digested 
ovalbumin was able to interact with the glutaraldehyde-fixed 
antigen-presenting cells, thereby activating ovalbumin- 
specific T H cells, whereas the native ovalbumin failed to do 
so. These results suggest that antigen processing involves the 
digestion of the protein into peptides that are recognized by 
the ovalbumin-specific T H cells. 



• 1ENTAL CONDITIONS 




' Experimental demonstration that antigen pro 

s (APCs) are fixed before exposure to antigen, they are unable 
ictivate T H cells, (b) In contrast, APCs fixed at least 1 h after 
gen exposure can activate T H cells, (c) When APCs are fixed 



before antigen exposure and incubated with peptide digests of the 
antigen (rather than native antigen), they also can activate T H cells. 
T H -cell activation is determined by measuring a specific T H -cell 
response (e.g., cytokine secretion). 



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49 AM Page 181 



8536d:Goldsby et al . / Immunology 5e-: 



Generation of B-Cell and T-Cell Respor 



Professional antigen-presenting cells Nonprofessional antigen-presenting cells 



Dendritic cells (several types) 
Macrophages 



Fibroblasts (skin) 
Glial cells (brain) 
Pancreatic beta cell.' 



Thymic epithelial cells 
Thyroid epithelial cells 
Vascular endothelial ce 



At about the same time, A. Townsend and his colleagues 
began to identify the proteins of influenza virus that were 
recognized by T c cells. Contrary to their expectations, they 
found that internal proteins of the virus, such as matrix 
and nucleocapsid proteins, were often recognized by T c 
cells better than the more exposed envelope proteins. 
Moreover, Townsend's work revealed that T c cells recog- 
nized short linear peptide sequences of the influenza pro- 
tein. In fact, when noninfected target cells were incubated 
in vitro with synthetic peptides corresponding to se- 
quences of internal influenza proteins, these cells could be 
recognized by T c cells and subsequently lysed just as well 
as target cells that had been infected with live influenza 
virus. These findings along with those presented in Figure 
8-3 suggest that antigen processing is a metabolic process 
that digests proteins into peptides, which can then be dis- 
played on the cell membrane together with a class I or class 
II MHC molecule. 

Most Cells Can Present Antigen with Class I 
MHC; Presentation with Class II MHC 
Is Restricted to APCs 

Since all cells expressing either class I or class II MHC mole- 
cules can present peptides to T cells, stricdy speaking they all 
could be designated as antigen-presenting cells. However, by 
convention, cells that display peptides associated with class I 
MHC molecules to CD8 T c cells are referred to as target cells; 
cells that display peptides associated with class II MHC mole- 
cules to CD4 T H cells are called antigen-presenting cells 
(APCs). This convention is followed throughout this text. 

A variety of cells can function as antigen-presenting cells. 
Their distinguishing feature is their ability to express class II 
MHC molecules and to deliver a co-stimulatory signal. Three 
cell types are classified as professional antigen-presenting 
cells: dendritic cells, macrophages, and B lymphocytes. These 
cells differ from each other in their mechanisms of antigen 
uptake, in whether they constitutively express class II MHC 
molecules, and in their co-stimulatory activity: 

■ Dendritic cells are the most effective of the antigen- 
presenting cells. Because these cells constitutively express 
a high level of class II MHC molecules and co- 
stimulatory activity, they can activate naive T H cells. 



■ Macrophages must be activated by phagocytosis of 
particulate antigens before they express class II MHC 
molecules or the co-stimulatory B7 membrane 
molecule. 

■ B cells constitutively express class II MHC molecules but 
must be activated before they express the co-stimulatory 
B7 molecule. 

Several other cell types, classified as nonprofessional 
antigen-presenting cells, can be induced to express class II 
MHC molecules or a co-stimulatory signal (Table 8-1). 
Many of these cells function in antigen presentation only 
for short periods of time during a sustained inflammatory 
response. 

Because nearly all nucleated cells express class I MHC 
molecules, virtually any nucleated cell is able to function as a 
target cell presenting endogenous antigens to T c cells. Most 
often, target cells are cells that have been infected by a virus 
or some other intracellular microorganism. However, altered 
self-cells such as cancer cells, aging body cells, or allogeneic 
cells from a graft can also serve as targets. 



Evidence for Two Processing 
and Presentation Pathways 

The immune system uses two different pathways to eliminate 
intracellular and extracellular antigens. Endogenous anti- 
gens (those generated within the cell) are processed in the cy- 
tosolic pathway and presented on the membrane with class I 
MHC molecules; exogenous antigens (those taken up by en- 
docytosis) are processed in the endocytic pathway and pre- 
sented on the membrane with class II MHC molecules 
(Figure 8-4). 

Experiments carried out by L. A. Morrison and T J. 
Braciale provided early evidence that the antigenic peptides 
presented by class I and class II MHC molecules are derived 
from different processing pathways. These researchers based 
their experimental protocol on the properties of two clones 
of T c cells, one that recognized influenza hemagglutinin 
(HA) associated with a class I MHC molecule, and an 
atypical T c line that recognized the same antigen associated 
with a class II MHC molecule. (In this case, and in some 



A 



:7 9 Mac 79:45_BWrf 



Antigen Processing and Presenta 



CYTOSOL1C PATHWAY 
± Ubiquitin 

Endogenous > Cytoplasmic - 

antigens proteasome 






-> Endocytic compartments 






endocyti 
moleculf 



Overview of cytosolic and endocytic pathways for 
g antigen. The proteasome complex contains enzymes 
e peptide bonds, converting proteins into peptides. The 
peptides from proteasome cleavage and those from 
compartments associate with class I or class II MHC 
, and the peptide-MHC complexes are then transported 




to the cell membrane. TAP (transporter of ontii 
transports the peptides to the endoplasmic reticuli 
noted that the ultimate fate of most peptides in th 
of these pathways, but rather to be degraded < 



c peptides) 
It should be 



others as well, the association of T-cell function with MHC 
restriction is not absolute). In one set of experiments, target 
cells that expressed both class I and class II MHC molecules 
were incubated with infectious influenza virus or with UV- 
inactivated influenza virus. (The inactivated virus retained 
its antigenic properties but was no longer capable of replicat- 
ing within the target cells.) The target cells were then incu- 
bated with the class I-restricted or the atypical class II- 
restricted T c cells and subsequent lysis of the target cells was 
determined. The results of their experiments, presented in 
Table 8-2, show that the class II-restricted T c cells responded 
to target cells treated with either infectious or noninfectious 
influenza virions. The class I-restricted T c cells responded 



only to target cells treated with infectious virions. Similarly, 
target cells that had been treated with infectious influenza 
virions in the presence of emetine, which inhibits viral pro- 
tein synthesis, stimulated the class II-restricted T c cells but 
not the class I-restricted T c cells. Conversely, target cells that 
had been treated with infectious virions in the presence of 
chloroquine, a drug that blocks the endocytic processing 
pathway, stimulated class I- but not class II-restricted T c 
cells. 

These results support the distinction between the process- 
ing of exogenous and endogenous antigens, including the 
preferential association of exogenous antigens with class II 
MHC molecules and of endogenous antigens with class I 



CTL ACTIVITY* 



Treatment of target cells'* 
Infectious virus 

UV-inactivated virus (noninfectious) 
Infectious virus + emetine 
Infectious virus + chloroquine 



Class I restricted 



with the indicated pr 



Class II restricted 



is of influenza virus and other agents. Emetine 



etermined by lysis (+) and no lysis (-] 
IURCE: Adapted from T. J. Braciale et al 



imunol. Rev. 98:95. 



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3 AM Page 190 mac79 Mac 7 9 :4 5_BW / rf 



Generation of B-Cell and T-Cell Respor 



MHC molecules. Association of viral antigen with class I 
MHC molecules required replication of the influenza virus 
and viral protein synthesis within the target cells; association 
with class II did not. These findings suggested that the pep- 
tides presented by class I and class II MHC molecules are 
trafficked through separate intracellular compartments; class 
I MHC molecules interact with peptides derived from cy- 
tosolic degradation of endogenously synthesized proteins, 
class II molecules with peptides derived from endocytic 
degradation of exogenous antigens. The next ( 
e these two pathways in detail. 



Endogenous Antigens: 
The Cytosolic Pathway 

In eukaryotic cells, protein levels are carefully regulated. 
Every protein is subject to continuous turnover and is de- 
graded at a rate that is generally expressed in terms of its half- 
life. Some proteins (e.g., transcription factors, cyclins, and 
key metabolic enzymes) have very short half-lives; dena- 
tured, misfolded, or otherwise abnormal proteins also are de- 
graded rapidly. The pathway by which endogenous antigens 
are degraded for presentation with class I MHC molecules 
utilizes the same pathways involved in the normal turnover 
of intracellular proteins. 

Peptides for Presentation Are Generated by 
Protease Complexes Called Proteasomes 

Intracellular proteins are degraded into short peptides by a cy- 
tosolic proteolytic system present in all cells. Those proteins 
targeted for proteolysis often have a small protein, called 
ubiquitin, attached to them (Figure 8-5a). Ubiquitin-protein 
conjugates can be degraded by a multifunctional protease 
complex called a proteasome. Each proteasome is a large 
(26S), cylindrical particle consisting of four rings of pro- 
tein subunits with a central channel of diameter 10-50 A. 
A proteasome can cleave peptide bonds between 2 or 3 
different amino acid combinations in an ATP-dependent 
process (Figure 8-5b). Degradation of ubiquitin-protein 
complexes is thought to occur within the central hollow of 
the proteasome. 

Experimental evidence indicates that the immune system 
utilizes this general pathway of protein degradation to 
produce small peptides for presentation with class I MHC 
molecules. The proteasomes involved in antigen processing 
include two subunits encoded within the MHC gene cluster, 
LMP2 and LMP7, and a third non-MHC protein, LMP10 
( also called MECL- 1 ) . All three are induced by increased lev- 
els of the T-cell cytokine IFN-7. The peptidase activities of 
proteasomes containing LMP2, LMP7, and LMP10 preferen- 
tially generate peptides that bind to MHC class I molecules. 
Such proteasomes, for example, show increased hydrolysis 
of peptide bonds that follow basic and/or hydrophobic 




■ Cytosolic proteolytic system for degradation of intra- 
r proteins, (a) Proteins to be degraded are often covalently 



inked tc 



:alled u 



this 



I protein 

quires ATP, a ubiquinating enzyme complex lin 
molecules to a lysine-amino group near the am 
protein, (b) Degradation of ubiquitin-protein 
within the central channel of proteasomes, ger 
peptides. Proteasomes are large cylindrical partii 
catalyze cleavage of peptide bonds. 



1, which re- 
inusofthe 



residues. As described 
class I MHC molecules 
drophobic or basic residi 



' 7, peptides that bind to 
lmost exclusively with hy- 



Peptides Are Transported from the Cytosol 
to the Rough Endoplasmic Reticulum 

Insight into the role that peptide transport, the delivery of 
peptides to the MHC molecule, plays in the cytosolic pro- 
cessing pathway came from studies of cell lines with defects 
in peptide presentation by class I MHC molecules. One such 
mutant cell line, called RMA-S, expresses about 5% of the 
normal levels of class I MHC molecules on its membrane. Al- 
though RMA-S cells synthesize normal levels of class I a 
chains and [^-microglobulin, neither molecule appears on 
the membrane. A clue to the mutation in the RMA-S cell line 
was the discovery by A. Townsend and his colleagues that 
"feeding" these cells peptides restored their level of mem- 
brane-associated class I MHC molecules to normal. These 
investigators suggested that peptides might be required to 
stabilize the interaction between the class I a chain and 
(3 2 -microglobulin. The ability to restore expression of class 
I MHC molecules on the membrane by feeding the cells 
predigested peptides suggested that the RMA-S cell line 
might have a defect in peptide transport. 



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3 AM Page 191 mac79 Mac 7 9 :4 5_BW / rf 



Antigen Processing and Presenta 



Subsequent experiments showed that the defect in the 
RMA-S cell line occurs in the protein that transports pep- 
tides from the cytoplasm to the RER, where class I molecules 
are synthesized. When RMA-S cells were transfected with a 
functional gene encoding the transporter protein, the cells 
began to express class I molecules on the membrane. The 
transporter protein, designated TAP (for transporter asso- 
ciated with antigen processing) is a membrane-spanning 
heterodimer consisting of two proteins: TAP1 and TAP2 
(Figure 8-6a). In addition to their multiple transmembrane 
segments, the TAP1 and TAP2 proteins each have a domain 
projecting into the lumen of the RER, and an ATP-binding 
domain that projects into the cytosol. Both TAP1 and TAP2 
belong to the family of ATP-binding cassette proteins found 
in the membranes of many cells, including bacteria; these 
proteins mediate ATP-dependent transport of amino acids, 
sugars, ions, and peptides. 

Peptides generated in the cytosol by the proteasome are 
translocated by TAP into the RER by a process that requires 
the hydrolysis of ATP (Figure 8-6b). TAP has the highest 
affinity for peptides containing 8-10 amino acids, which is 
the optimal peptide length for class I MHC binding. In addi- 
tion, TAP appears to favor peptides with hydrophobic or ba- 
sic carboxyl-terminal amino acids, the preferred anchor 
residues for class I MHC molecules. Thus, TAP is optimized 
to transport peptides that will interact with class I MHC 
molecules. 

The TAP1 and TAP2 genes map within the class II MHC 
region, adjacent to the LMP2 and LMP7 genes (see Figure 
7-15). Both the transporter genes and these LMP genes are 
polymorphic; that is, different allelic forms of these genes 
exist within the population. Allelic differences in LMP-me- 
diated proteolytic cleavage of protein antigens or in the 
transport of different peptides from the cytosol into the RER 
may contribute to the observed variation among individuals 
in their response to different endogenous antigens. TAP 
deficiencies can lead to a disease syndrome that has aspects 
of both immunodeficiency and autoimmunity (see Clinical 
Focus). 

Peptides Assemble with Class I MHC Aided 
by Chaperone Molecules 

Like other proteins, the a chain and (^-microglobulin 
components of the class I MHC molecule are synthesized 
on polysomes along the rough endoplasmic reticulum. As- 
sembly of these components into a stable class I MHC 
molecular complex that can exit the RER requires the 
presence of a peptide in the binding groove of the class I 
molecule. The assembly process involves several steps and 
includes the participation of molecular chaperones, which 
facilitate the folding of polypeptides. The first molecular 
chaperone involved in class I MHC assembly is calnexin, a 
resident membrane protein of the endoplasmic reticulum. 
Calnexin associates with the free class I a chain and pro- 
motes its folding. When (^-microglobulin binds to the a 
chain, calnexin is released and the class I molecule associ- 



C CATP) ^ ' CATP) ) 

) C Cytosol 



RER membrane 



^-fifl^=^ 




Generation of antigenic peptide-class I MHC com- 
plexes in the cytosolic pathway, (a) Schematic diagram of TAP, a het- 
erodimer anchored in the membrane of the rough endoplasmic 
reticulum (RER). The two chains are encoded by TAP1 and TAP2. The cy- 
tosolic domain in each TAP subunit contains an ATP-binding site, and 
peptide transport depends on the hydrolysis of ATP. (b) In the cytosol, 
association of LMP2, LMP7, and LMP10 (black spheres) with a protea- 
some changes its catalytic specificity to favor production of peptides that 
bind to class I MHC molecules. Within the RER membrane, a newly syn- 
thesized class I a chain associates with calnexin until (3 2 -microglobulin 
binds to the a chain. The class I a chain/£> 2 -microglobulin heterodimer 
then binds to calreticulin and the TAP-associated protein tapasin. When 
a peptide delivered by TAP is bound to the class I molecule, folding of 
MHC class I is complete and it is released from the RER and transported 
through the Colgi to the surface of the cell. 



ates with the chaperone calreticulin and with tapasin. 
Tapasin (TAP-associated protein) brings the TAP trans- 
porter into proximity with the class I molecule and 
allows it to acquire an antigenic peptide (Figure 8-7). The 
physical association of the a chain-(3 2 -microglobulin 



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49 AM Page 192 n 



8536d:Goldsby et al . / Immunology 5e-: 



Generation of B-Cell and T-Cell Respor 



CLINICAL FOCUS 



Deficiency in Transporters 
Associated with Antigen 
Presentation (TAP) Leads to a 
Diverse Disease Spectrum 



tiVel aeon 
dition known as bare lymphocyte syn- 
drome, or BLS, has been recognized for 
more than 22 years. The lymphocytes in 
BLS patients express MHC molecules at 

not at all. In type 1 BLS, a deficiency in 
MHC class I molecules exists; in type 2 
BLS, expression of class II molecules is 
impaired. The pathogenesis of one type 
of BLS underscores the importance of 
the class I family of MHC molecules in 
their dual roles of preventing autoim- 
munity as well as defending against 
pathogens. 

Defects in promoter sequences that 
preclude MHC gene transcription were 
found for some type 2 BLS cases, but in 



lany 



the nature of the under 



lying defect is not known. A recent study 
has identified a group of patients with 
type 1 BLS due to defects in TAP! or 
TAP2 genes. Manifestations of the TAP 
deficiency were consistent in this patient 
group and define a unique disease. As 
described earlier in this chapter, TAP pro- 
teins are necessary for the loading of 
peptides onto class I molecules, a step 
that is essential for expression of class I 
MHC molecules on the cell surface. Lym- 
phocytes in individuals with TAP defi- 



ciency express le 
significantly lowi 



s of class I molec 



jntrols. 



creased numbers of Nl< and 78 T cells, 
and decreased levels of CD8 + a|3 T cells. 
As we shall see, the disease manifesta- 
tions are reasonably well explained by 
these deviations in the levels of certain 
cells involved in immune function. 

In early life the TAP-deficient individ- 
ual suffers frequent bacterial infections 



of the upper respiratory tract, and in the 
second decade begins to have chronic in- 
fection of the lungs. It is thought that a 
post-nasal-drip syndrome common in 
younger patients promotes the bacterial 
lung infections in later life. Noteworthy is 
the absence of any severe viral infection, 
which is common in immunodeficien- 
cies with T-cell involvement (see Chapter 
19). Bronchiectasis (dilation of the 
bronchial tubes) often occurs and recur- 



n lead to lung damage 



n the extrem- 



ring infectioi 

that may be fatal. The rr 

mark of the deficiency is the 

of necrotizing skin 

ities and the midface. These 

cerate and may cause disfigur 

figure). The skin lesions are probably due 

to activated NK cells and 78 T cells; NK 



cells were isolated from biopsied skin 
from several patients, supporting this 
possibility. Normally, the activity of NK 
cells is limited through the action of 
killer-cell-inhibitory receptors (KIRs), 
which deliver a negative signal to the NK 
cell following interaction with class I 
molecules (see Chapter 14). The defi- 
ciency of class I molecules in TAP-related 
BLS patients explains the excessive activ- 
ity of the NK cells. Activation of NK cells 
further explains the absence of severe 
virus infections, which are limited by NK 
and 78 cells. 

The best treatment for the character- 
istic lung infections appears to be antibi- 
otics and intravenous immunoglobulin. 
Attempts to limit the skin disease by im- 
munosuppressive regimens, such as 
steroid treatment or cytotoxic agents, 
can lead to exacerbation of lesions and is 
therefore contraindicated. Mutations in 
the promoter region of TAPthat preclude 
expression of the gene were found for 
several patients, suggesting the possibil- 
ity of gene therapy, but the cellular distri- 
bution of class I is so widespread that it 
is not clear what cells would need to be 
corrected to alleviate all symptoms. 




Necrotizing granulomatous lesions in the midface of patient with TAP-deficiency syn- 
drome. TAP deficiency leads to a condition with symptoms characteristic of autoimmu- 
nity, such as the skin lesions that appear on the extremities and the midface, as well as 
immunodeficiency that causes chronic sinusitis, leading to recurrent lung infection. 
[From S. D. Cadola et al., 1999, Lancet 354:1598, and 2000, Clinical and Experimental 
Immunology 121:173.] 



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Antigen Processing and Presenta 




Tapasin Call 



I Assembly and 
cules. Newly formed class I , 
molecular chaperone, in the RER 
to p 2 -microglobulin releases call 



chai 



mbrane. Subsequent binding 









chaperonin calreticulin and to tapasin 
peptide transporter TAP. This association p 
antigenic peptide, which stabilizes the cla: 
complex, allowing its release from the RER. 





Calreticulin 




ciated 


with the 


romotes 


bind 


ngofan 


ss 1 mo 


ecule 


-peptide 



heterodimer with the TAP protein (see Figure 8-6b) pro- 
motes peptide capture by the class I molecule before the pep- 
tides are exposed to the luminal environment of the RER. 
Peptides not bound by class I molecules are rapidly degraded. 
As a consequence of peptide binding, the class I molecule dis- 
plays increased stability and can dissociate from calreticulin 
and tapasin, exit from the RER, and proceed to the cell sur- 
face via the Golgi. An additional chaperone protein, ERp57, 
has been observed in association with calnexin and calretic- 
ulin complexes. The precise role of this resident endoplasmic 
reticulum protein in the class I peptide assembly and loading 
process has not yet been defined, but it is thought to con- 
tribute to the formation of disulfide bonds during the matu- 
ration of class I chains. Because its role is not clearly defined, 
ERp57 is not shown in Figures 8-6 and 8-7. 



Exogenous Antigens: The Endocytic 
Pathway 

Figure 8-8 recapitulates the endogenous pathway discussed 
previously (left side), and compares it with the separate exoge- 
nous pathway (right), which we shall now consider. Whether 
an antigenic peptide associates with class I or with class II mol- 
ecules is dictated by the mode of entry into the cell, either ex- 
ogenous or endogenous, and by the site of processing. 

Antigen-presenting cells can internalize antigen by phago- 
cytosis, endocytosis, or both. Macrophages internalize antigen 
by both processes, whereas most other APCs are not phago- 
cytic or are poorly phagocytic and therefore internalize exoge- 
nous antigen only by endocytosis (either receptor-mediated 
endocytosis or pinocytosis). B cells, for example, internalize 
antigen very effectively by receptor-mediated endocytosis us- 
ing antigen-specific membrane antibody as the receptor. 

Peptides Are Generated from Internalized 
Molecules in Endocytic Vesicles 

Once an antigen is internalized, it is degraded into peptides 
within compartments of the endocytic processing pathway. As 



the experiment shown in Figure 8-3 demonstrated, internal- 
ized antigen takes 1-3 h to transverse the endocytic pathway 
and appear at the cell surface in the form of peptide-class II 
MHC complexes. The endocytic pathway appears to involve 
three increasingly acidic compartments: early endosomes (pH 
6.0-6.5); late endosomes, or endolysosomes (pH 5.0-6.0); and 
lysosomes (pH 4.5-5.0). Internalized antigen moves from 
early to late endosomes and finally to lysosomes, encountering 
hydrolytic enzymes and a lower pH in each compartment (Fig- 
ure 8-9). Lysosomes, for example, contain a unique collection 
of more than 40 acid-dependent hydrolases, including pro- 
teases, nucleases, glycosidases, lipases, phospholipases, and 
phosphatases. Within the compartments of the endocytic 
pathway, antigen is degraded into oligopeptides of about 13- 
1 8 residues, which bind to class II MHC molecules. Because the 
hydrolytic enzymes are optimally active under acidic condi- 
tions (low pH), antigen processing can be inhibited by chemi- 
cal agents that increase the pH of the compartments (e.g., 
chloroquine) as well as by protease inhibitors (e.g., leupeptin). 
The mechanism by which internalized antigen moves 
from one endocytic compartment to the next has not been 
conclusively demonstrated. It has been suggested that early 
endosomes from the periphery move inward to become late 
endosomes and finally lysosomes. Alternatively, small trans- 
port vesicles may carry antigens from one compartment to 
the next. Eventually the endocytic compartments, or por- 
tions of them, return to the cell periphery, where they fuse 
with the plasma membrane. In this way, the surface receptors 
are recycled. 

The Invariant Chain Guides Transport 
of Class II MHC Molecules 
to Endocytic Vesicles 

Since antigen-presenting cells express both class I and class II 
MHC molecules, some mechanism must exist to prevent 
class II MHC molecules from binding to the same set of anti- 
genic peptides as the class I molecules. When class II MHC 
molecule are synthesized within the RER, three pairs of class 
II a(3 chains associate with a preassembled trimer of a 



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Generation of B-Cell and T-Cell Respor 



VISUALIZING CONCEPTS 




for endogenous (gn 



'ate antigen-presenting pathways are utilized 
»n) and exogenous (red) antigens. The mode 
cells and the site of antigen processing de- 



termine whether antigenic peptides associate with class I MHC 
molecules in the rough endoplasmic reticulum or with class II 
molecules in endocytic compartments. 



protein called invariant chain (Ii, CD74). This trimeric pro- 
tein interacts with the peptide-binding cleft of the class II 
molecules, preventing any endogenously derived peptides 



from binding to the cleft while the class II molecule is within 
the RER (see right side of Figure 8-8). The invariant chain 
also appears to be involved in the folding of the class II a and 



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Antigen Processing and Presenta 




Lysosome 
Lateendosome pH 4 . 5-5.0 



A nonclassical class II MHC molecule called HLA-DM is 
required to catalyze the exchange of CLIP with antigenic 
peptides (Figure 8- 10a). MHC class II genes encoding HLA- 
DM have been identified in the mouse and rabbit, indicating 






V ♦ 1—1 



^Released CLIP 

CLIP o it »-Peptides 

"I J HLA-DM 



^fv» 



I Generation of antigenic peptides in the endocytic 
processing pathway. Internalized exogenous antigen moves through 
several acidic compartments, in which it is degraded into peptides 
that ultimately associate with class II MHC molecules transported in 
vesicles from the Colgi complex. The cell shown here is a B cell, 
which internalizes antigen by receptor-mediated endocytosis, with 
the membrane-bound antibody functioning as an antigen-specific 



(3 chains, their exit from the RER, and the subsequent routing 
of class II molecules to the endocytic processing pathway 
from the trans-Golgi network. 

The role of the invariant chain in the routing of class II mol- 
ecules has been demonstrated in transfection experiments with 
cells that lack the genes encoding class II MHC molecules and 
the invariant chain. Immunofluorescent labeling of such cells 
transfected only with class II MHC genes revealed class II mol- 
ecules localized within the Colgi complex. However, in cells 
transfected with both the class II MHC genes and invariant- 
chain gene, the class II molecules were localized in the cytoplas- 
mic vesicular structures of the endocytic pathway. The 
invariant chain contains sorting signals in its cytoplasmic tail 
that directs the transport of the class II MHC complex from the 
trans-Golgi network to the endocytic compartments. 

Peptides Assemble with Class II MHC 
Molecules by Displacing CLIP 

Recent experiments indicate that most class II MHC-invari- 
ant chain complexes are transported from the RER, where 
they are formed, through the Colgi complex and trans-Golgi 
network, and then through the endocytic pathway, moving 
from early endosomes to late endosomes, and finally to lyso- 
somes. As the proteolytic activity increases in each successive 
compartment, the invariant chain is gradually degraded. 
However, a short fragment of the invariant chain termed 
CLIP (for class II-associated invariant chain peptide) remains 
bound to the class II molecule after the invariant chain has 
been cleaved within the endosomal compartment. CLIP 
physically occupies the peptide-binding groove of the class II 
MHC molecule, presumably preventing any premature bind- 
ing of antigenic peptide (see Figure 8-8). 




cules. Within the 
d class II MHC 

II molecule and 



5. Digestion of the invariant 
ining in the binding groove 
i nonclassical MHC class II 
impartments, mediates ex- 
;al class II mol- 

role in the dis- 



assembly of class II MHCrr 
rough endoplasmic reticulum, a newly synthe 
molecule binds an invariant chain. The bound 
vents premature binding of peptides to the cl 
helps to direct the complex to endocytic comp 
peptides derived from exogenous antige 
chain leaves CLIP, a small fragment rerr 
of the class II MHC molecule. HLA-DM, a r 
molecule expressed within endosomal con 
change of antigenic peptides for CLIP. The 
ecule H LA-DO may act as a negative reg 
processing by binding to HLA-DM and inr 
sociation of CLIP from class II molecules, (b) Comparison of three- 
dimensional structures showing the binding groove of HLA class II 
molecules (a1 and (31) containing different antigenic peptides or 
CLIP. The red lines show DR4 complexed with collagen II peptide, 
yellow lines are DR1 with influenza hemagglutinin peptide, and blue 
lines are DR3 associated with CLIP. (N indicates the amino terminus 
and C the carboxyl terminus of the peptides.) No major differences in 
the structures of the class II molecules or in the conformation of the 
bound peptides are seen. This comparison shows that CLIP binds 
the class II molecule in a manner identical to that of antigenic pep- 
tides. [Part (b)from Dessen et at., 1997, Immunity 7:473-481; cour- 
tesy of Don Wiley, Harvard University.] 



A 



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Generation of B-Cell and T-Cell Respor 



that HLA-DM is widely conserved among mammalian 
species. Like other class II MHC molecules, HLA-DM is a 
heterodimer of a and (3 chains. However, unlike other class II 
molecules, HLA-DM is not polymorphic and is not ex- 
pressed at the cell membrane but is found predominantly 
within the endosomal compartment. The DMa and DM/3 
genes are located near the TAP and IMP genes in the MHC 
complex of humans and DM is expressed in cells that express 
classical class II molecules. 

The reaction between HLA-DM and the class II CLIP 
complex facilitating exchange of CLIP for another peptide is 
impaired in the presence of HLA-DO, which binds to HLA- 
DM and lessens the efficiency of the exchange reaction. HLA- 
DO, like HLA-DM, is a nonclassical and nonpolymorphic 
class II molecule that is also found in the MHC of other 
species. HLA-DO differs from HLA-DM in that it is ex- 
pressed only by B cells and the thymus, and unlike other class 
II molecules, its expression is not induced by IFN-7. An ad- 
ditional difference is that the genes encoding the a and the (3 
chains of HLA-DO are not adjacent in the MHC as are all 
other class II a and (3 pairs (see Fig 7-15). 

An HLA-DR3 molecule associated with CLIP was isolated 
from a cell line that did not express HLA-DM and was there- 
fore defective in antigen processing. Superimposing the 
structure of HLA-DR3-CLIP on another DR molecule 
bound to antigenic peptide reveals that CLIP binds to class II 
in the same stable manner as the antigenic peptide (Figure 8- 
10b). The discovery of this stable complex in a cell with de- 
fective HLA-DM supports the argument that HLA-DM is 
required for the replacement of CLIP. 

Although it certainly modulates the activity of HLA-DM, 
the precise role of HLA-DO remains obscure. One possibility 
is that it acts in the selection of peptides bound to class II 
MHC molecules in B cells. DO occurs in complex with DM 
in these cells and this association continues in the endosomal 
compartments. Conditions of higher acidity weaken the as- 
sociation of DM/DO and increase the possibility of antigenic 
peptide binding despite the presence of DO. Such a pH-de- 
pendent interaction could lead to preferential selection of 
class II-bound peptides from lysosomal compartments in B 
cells as compared with other APCs. 

As with class I MHC molecules, peptide binding is required 
to maintain the structure and stability of class II MHC mole- 
cules. Once a peptide has bound, the peptide-class II complex 
is transported to the plasma membrane, where the neutral pH 
appears to enable the complex to assume a compact, stable 
form. Peptide is bound so strongly in this compact form that it 
is difficult to replace a class II-bound peptide on the mem- 
brane with another peptide at physiologic conditions. 



Presentation of Nonpeptide Antigens 

To this point the discussion has been limited to peptide anti- 
gens and their presentation by classical class I and II MHC 
molecules. It is well known that nonprotein antigens also are 



recognized by the immune system, and there are reports dat- 
ing back to the 1980s of T cell proliferation in the presence of 
nonprotein antigens derived from infectious agents. More re- 
cent reports indicate that T cells that express the 78 TCR (T- 
cell receptors are dimers of either a (3 or 78 chains) that react 
with glycolipid antigens derived from bacteria such as My- 
cobacterium tuberculosis. These nonprotein antigens are pre- 
sented by members of the CD1 family of nonclassical class I 
molecules. 

The CD1 family of molecules associates with (^-mi- 
croglobulin and has general structural similarity to class I 
MHC molecules. There are five genes encoding human CD1 
molecules (CD1A-E, encoding the gene products CDla-d, 
with no product yet identified for E). These genes are located 
not within the MHC but on chromosome 1 (Figure 8-1 la). 
The genes are classified into two groups based on sequence 
homology. Group 1 includes CD1A, B, C, and E; CD1D is in 
group 2. All mammalian species studied have CD1 genes, al- 
though the number varies. Rodents have only group 2 CD1 
genes, the counterpart of human CD1D, whereas rabbits, like 
humans, have five genes, including both group 1 and 2 types. 
Sequence identity of CD1 with classical class I molecules is 
considerably lower than the identity of the class I molecules 
with each other. Comparison of the three-dimensional struc- 
ture of the mouse CDldl with the class I MHC molecule H- 
2k shows that the antigen-binding groove of the CDldl 
molecules is deeper and more voluminous than that of the 
classical class I molecule (Fig 8-1 lb). 

Expression of CD1 molecules varies according to subset; 
CD1D1 genes are expressed mainly in nonprofessional APCs 
and on certain B-cell subsets. The mouse CDldl is more 
widely distributed and found on T cells, B cells, dendritic 
cells, hepatocytes, and some epithelial cells. The CD1A, B, 
and C genes are expressed on immature thymocytes and pro- 
fessional APCs, mainly those of the dendritic type. CD1C 
gene expression is seen on B cells, whereas the CD1A and B 
products are not. CD1 genes can be induced by exposure to 
certain cytokines such as GM-CSF or IL-3. The intracellular 
trafficking patterns of the CD1 molecules differ; for example, 
CD la is found mostly in early endosomes or on the cell sur- 
face; CDlb and CDld localize to late endosomes; and CDlc 
is found throughout the endocytic system. 

Certain CD1 molecules are recognized by T cells in the ab- 
sence of foreign antigens, and self restriction can be demon- 
strated in these reactions. Examination of antigens presented 
by CD1 molecules revealed them to be lipid components 
(mycolic acid) of the M. tuberculosis cell wall. Further studies 
of CD 1 presentation indicated that a glycolipid (lipoarabino- 
mannan) from Mycobacterium leprae could also be presented 
by these molecules. The data concerning CD1 antigen pre- 
sentation point out the existence of a third pathway for the 
processing of antigens, a pathway with distinct intracellular 
steps that do not involve the molecules found to facilitate 
class I antigen processing. For example, CD1 molecules are 
able to process antigen in TAP-deficient cells. Recent data 
indicate that the CD la and lb molecules traffic differently, 



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(a) HUMAN CHROMOSOME 1 



Antigen Processing and Presenta 



lie: CD1D 



CD1A CD1C CD1B CD1E 



MOUSE CHROMOSOME 3 

— H — 

Gene name: CD1D1 CD1D2 




| The CDI family of genes and structure of a CDId 
molecule, (a) The genes encoding the CD1 family of molecules in 
human (top) and mouse (bottom). The genes are separated into 
two groups based on sequence identity; CDIA, B, C, and E are 
group 1 , CDI D genes are group 2. The products of the pink genes 
have been identified; products of grey genes have not yet been 



detected, (b) Comparison of the crystal structures of mouse non- 
classical CD1 and classical class I molecule H-2fc b . Note the differ- 
ences in the antigen binding grooves. [Part (b) reprinted from 
Trends in Immunology (formerly Immunology TodayJ, Vol. 19, S. A. 
Porcelli and R. L Modlin, The CDI family of lipid antigen presenting 
molecules, pp. 362-368, 1998, with permission from Elsevier Science.] 



with CD la at the surface or in the recycling endocytic 
compartments and CDlb and CDId in the lysomal compart- 
ments. Exactly how the CD 1 pathway complements or inter- 
sects the better understood class I and class II pathways remains 
an open question. The T-cell types reactive to CDI were first 
thought to be limited to T cells expressing the 78 TCR and lack- 
ing both CD4 and CD8, or T cells with a single TCR a chain, 
but recent reports indicate that a wider range of T-cell types will 
recognize CDI -presenting cells. Recent evidence indicates that 
natural killer T cells recognize CD Id molecules presenting au- 
tologous antigen. This may represent a mechanism for elimi- 
nating cells that are altered by stress, senescence, or neoplasia. 



1 T-cells recognize antigen displayed within the cleft of a 
self-MHC molecule on the membrane of a cell. 

1 In general, CD4 + T H cells recognize antigen with class II 
MHC molecules on antigen-processing cells. 



1 CD8 T c cells recognize antigen with class I MHC mole- 






it cells. 



1 Complexes between antigenic peptides and MHC mole- 
cules are formed by degradation of a protein antigen in 
one of two different antigen-processing pathways. 

1 Endogenous antigens are degraded into peptides within 
the cytosol by proteasomes and assemble with class I mol- 
ecules in the RER. 

1 Exogenous antigens are internalized and degraded within 
the acidic endocytic compartments and subsequently pair 
with class II molecules. 

1 Peptide binding to class II molecules involves replacing 
a fragment of invariant chain in the binding cleft by 
a process catalyzed by nonclassic MHC molecule 
HLA-DM. 

1 Presentation of nonpeptide (lipid and glycolipid) anti- 
gens derived from bacteria involves the class I— like CDI 
molecules. 



www.whfreeman.ci 
and quiz of key ter 



ft 5 



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Generation of B-Cell and T-Cell Respor 



References 

Alfonso, C, and L. Karlsson. 2000. Nonclassical class II mole- 
cules. Ann. Rev. Immunol. 18:113. 

Brodsky, F. M., et al. 1999. Human pathogen subversion of anti- 
gen presentation. Immunol. Reviews. 168:199. 

Busch, R., et al. 2000. Accessory molecules for MHC class II pep- 
tide loading. Curr. Opinion in Immunol. 12:99. 

Doherty, P. C, and R. M. Zinkernagel. 1975. H-2 compatibility is 
required for T-cell mediated lysis of target cells infected with 
lymphocytic choriomeningitis virus. /. Exp. Med. 141:502. 

Gadola, S. D., et al. 2000. TAP deficiency syndrome. Clin. Exp. 
Immunol. 121:173. 

Ghosh P., M. Amaya, E. Mellins, and D. C. Wiley. 1995. The 
structure of an intermediate in class II MHC maturation: CLIP 
bound to HLA-DR3. Nature 378:457. 

Jayawardena-Wolf, J., and A. Bendelac. 2001. CD1 and lipid anti- 
gens: intracellular pathways for antigen presentation. Curr. 
Opinions in Immunol. 13:109. 

Matsuda J. L., and M. Kroneberg. 2001. Presentation of self and 
microbial lipids by CD1 molecules. Curr. Opinion in Immunol. 
13:19. 

Ortmann, B., et al. 1997. A critical role for tapasin in the assem- 
bly and function of multimeric MHC class I-TAP complexes. 
Science 277:1306. 

Pamer, E., and P. Cresswell. 1998. Mechanisms of MHC class I- 
restricted antigen processing. Annu. Rev. Immunol. 16:323. 

Porcelli, S. A., and R. L. Modlin. 1999. The CD1 System: Antigen- 
presenting molecules for T-cell recognition of lipids and gly- 
colipids. Ann. Rev. Immunol. 17:297. 

Roche, P. A. 1999. Intracellular protein traffic in lymphocytes: 
"How do I get there from here?" Immunity 11:391. 

Van Ham, M., et al. 2000. What to do with HLA-DO? Immuno- 
genetics 51:765. 

Yewdell, J. W. 200 1 . Not such a dismal science: The economics of 
protein synthesis, folding, degradation, and antigen process- 
ing. Trends in Cell Biol. 11: 294 



Study Questions 



Clinical Focus Question Patients with TAP deficiency have 
partial immunodeficiency as well as autoimmune manifesta- 
tions. How do the profiles for patients' immune cells explain the 
partial immunodeficiency? Why is it difficult to design a gene 
therapy treatment for this disease, despite the fact that a single 
gene defect is implicated? 



Explain the difference between the terms antigen-presenting 
cell and target cell, as they are commonly used in immunology. 



2. Define the following term 

a. Self-MHC restriction 

b. Antigen processing 

c. Endogenous antigen 

d. Exogenous antigen 



!. L. A. Morrison and T J. Braciale conducted an experiment to 
determine whether antigens presented by class I or II MHC 
molecules are processed in different pathways. Their results 
are summarized in Table 8-2. 

a. Explain why the class I-restricted T c cells did not re- 
spond to target cells infected with UV- inactivated in- 

b. Explain why chloroquine inhibited the response of the 
class II-restricted T c cells to live virus. 

c. Explain why emetine inhibited the response of class I- 
restricted but not class II-restricted T c cells to live 



4. For each of the following cell components or processes, indi- 
cate whether it is involved in the processing and presentation 
of exogenous antigens (EX), endogenous antigens (EN), or 
both (B). Briefly explain the function of each item. 

a. Class I MHC molecules 

b. Class II MHC molecules 

c. Invariant (Ii) chains 

d. Lysosomal hydrolases 

e. TAP1 and TAP2 proteins 

f. Transport of vesicles from the RER to the Golgi 

complex 

g. Proteasomes 



i. Calnexin 

j. CLIP 

k. Tapasin 

5. Antigen-presenting cells have been shown to present 
lysozyme peptide 46-61 together with the class II IA mole- 
cule. When CD4 + T H cells are incubated with APCs and native 
lysozyme or the synthetic lysozyme peptide 46-61, T H -cell 



a. If chloroquine is added to the incubation mixture, presen- 
tation of the native protein is inhibited, but the peptide 
continues to induce T H -cell activation. Explain why this 

b. If chloroquine addition is delayed for 3 h, presentation of 
the native protein is not inhibited. Explain why this occurs. 

6. Cells that can present antigen to T H cells have been classified 
into two groups — professional and nonprofessional APCs. 

a. Name the three types of professional APCs. For each type 

- whether it expresses class II MHC molecules and a 
co-stimulatory signal constitutively or must be activated 
before doing so. 

b. Give three examples of nonprofessional APCs. When are 
these cells most likely to function in antigen presentation? 

7. Predict whether T H -cell proliferation or CTL-mediated cytol- 
ysis of target cells will occur with the following mixtures of 
cells. The CD4 + T H cells are from lysozyme-primed mice, and 
the CD8 + CTLs are from influenza-infected mice. Use R to 
indicate a response and NR to indicate no response. 

a. H-2 T H cells + lysozyme-pulsed H-2 

macrophages 

b. H-2* T H cells + lysozyme-pulsed ¥L-2 b/k 

macrophages 



A 



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Antigen Processing and Presenta 



c. H-2*T H cells + lysozyme-primed H-2 rf 

macrophages 
d. H-2* CTLs + influenza-infected H-2* 

macrophages 

e. H-2* CTLs + influenza-infected H-2 rf 

macrophages 

f. H-2 d CTLs + influenza-infected H-2 d/k 

macrophages 

. HLA-DM and HLA-DO are termed nonclassical MHC class II 
molecules. How do they differ from the classical MHC class 
II? How do they differ from each other? 



;ntly shown to present 



I. Molecules of the CD1 family w 
nonpeptide antigens. 

a. What is a major source of nonpeptide antigens? 

b. Why are CD1 molecules not classified as members of 
the MHC family even though they associate with (3 2 - 
microglobulin? 

c. What evidence suggests that the CD1 pathway is different 
from that utilized by classical class I MHC molecules? 



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T-Cell Receptor 




HE ANTIGEN-SPECIFIC NATURE OF T-CELL RESPONSES 

clearly implies that T cells possess an antigen- 
specific and clonally restricted receptor. However, 
the identity of this receptor remained unknown long after 
the B-cell receptor (immunoglobulin molecule) had been 
identified. Relevant experimental results were contradictory 
and difficult to conceptualize within a single model because 
the T-cell receptor (TCR) differs from the B-cell antigen- 
binding receptor in important ways. First, the T-cell receptor 
is membrane bound and does not appear in a soluble form 
as the B-cell receptor does; therefore, assessment of its struc- 
ture by classic biochemical methods was complicated, and 
complex cellular assays were necessary to determine its speci- 
ficity. Second, most T-cell receptors are specific not for anti- 
gen alone but for antigen combined with a molecule encoded 
by the major histocompatibility complex (MHC). This prop- 
erty precludes purification of the T-cell receptor by simple 
antigen-binding techniques and adds complexity to any ex- 
perimental system designed to investigate the receptor. 

A combination of immunologic, biochemical, and 
molecular-biological manipulations has overcome these 
problems. The molecule responsible for T-cell specificity 
was found to be a heterodimer composed of either a and p 
or 7 and 8 chains. Cells that express TCRs have approxi- 
mately 10 5 TCR molecules on their surface. The genomic 
organization of the T-cell receptor gene families and the 
means by which the diversity of the component chains is 
generated were found to resemble those of the B-cell re- 
ceptor chains. Further, the T-cell receptor is associated on 
the membrane with a signal-transducing complex, CD3, 
whose function is similar to that of the Ig-a/Ig-(3 complex 
of the B-cell receptor. 

Important new insights concerning T-cell receptors have 
been gained by recent structure determinations using x-ray 
crystallography, including new awareness of differences in 
how TCRs bind to class I or class II MHC molecules. This 
chapter will explore the nature of the T-cell receptor mole- 
cules that specifically recognize MHC-antigen complexes, as 
well as some that recognize native antigens. 



Early Studies of the T-Cell Receptor 

By the early 1980s, investigators had learned much about 
T-cell function but were thwarted in their attempts to 



ART TO COME 



■ Early Studies of the T-Cell Receptor 

■ a|3 and 78 T-Cell Receptors: Structure and Roles 

■ Organization and Rearrangement of TCR Genes 

■ T-Cell Receptor Complex: TCR-CD3 

■ T-Cell Accessory Membrane Molecules 

■ Three-Dimensional Structures of TCR-Peptide- 
MHC Complexes 

■ Alloreactivity of T Cells 



identify and isolate its antigen-binding receptor. The obvi- 
ous parallels between the recognition functions of T cells 
and B cells stimulated a great deal of experimental effort to 
take advantage of the anticipated structural similarities be- 
tween immunoglobulins and T-cell receptors. Reports 
published in the 1970s claimed discovery of immunoglob- 
ulin isotypes associated exclusively with T cells (IgT) and 
of antisera that recognize variable-region markers (idio- 
types) common to antibodies and T-cell receptors with 
similar specificity. These experiments could not be repro- 
duced and were proven to be incorrect when it was demon- 
strated that the T-cell receptor and immunoglobulins do 
not have common recognition elements and are encoded 
by entirely separate gene families. As the following sections 
will show, a sequence of well-designed experiments using 
cutting-edge technology was required to correctly answer 
questions about the structure of the T-cell receptor, the 
genes that encode it, and the manner in which it recognizes 
antigen. 



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' Immunology 5e: 



I 



Classic Experiments Demonstrated the 
Self-MHC Restriction oftheT-Cell Receptor 

By the early 1970s, immunologists had learned to generate 
cytotoxic T lymphocytes (CTLs) specific for virus-infected 
target cells. For example, when mice were infected with lym- 
phocytic choriomeningitis (LCM) virus, they would produce 
CTLs that could lyse LCM-infected target cells in vitro. Yet 
these same CTLs failed to bind free LCM virus or viral anti- 
gens. Why didn't the CTLs bind the virus or viral antigens di- 
rectly as immunoglobulins did? The answer began to emerge 
in the classic experiments of R. M. Zinkernagel and P. C. 
Doherty in 1974 (see Figure 8-2). These studies demon- 
strated that antigen recognition by T cells is specific not for 
viral antigen alone but for antigen associated with an MHC 
molecule (Figure 9-1). T cells were shown to recognize anti- 
gen only when presented on the membrane of a cell by a self- 
MHC molecule. This attribute, called self-MHC restriction, 
distinguishes recognition of antigen by T cells and B cells. In 
1996, Doherty and Zinkernagel were awarded the Nobel 
Prize for this work. 

Two models were proposed to explain the MHC restric- 
tion of the T-cell receptor. The dual-receptor model envi- 
sioned a T cell with two separate receptors, one for antigen 
and one for class I or class II MHC molecules. The altered-self 
model proposed that a single receptor recognizes an alter- 
ation in self-MHC molecules induced by their association 
with foreign antigens. The debate between proponents of 
these two models was waged for a number of years, until an 
elegant experiment by J. Kappler and P. Marrack demon- 
strated that specificity for both MHC and antigen resides in a 
single receptor. An overwhelming amount of structural and 
functional data has since been added in support of the 
altered-self model. 




► Self-MHC restriction of the T-cell receptor (TCR). A 
particular TCR is specific for both an antigenic peptide and a self- 
MHC molecule. In this example, the H^CTL is specific for viral pep- 
tide A presented on an H-2* c target cell (a). Antigen recognition does 
not occur when peptide B is displayed on an U-2 k target cell (b) nor 
when peptide A is displayed on an H-2 d target cell (c). 



T-Cell Receptors Were Isolated by Using 
Clonotypic Antibodies 

Identification and isolation of the T-cell receptor was accom- 
plished by producing large numbers of monoclonal antibod- 
ies to various T-cell clones and then screening the antibodies 
to find one that was clone specific, or clonotypic. This ap- 
proach assumes that, since the T-cell receptor is specific for 
both an antigen and an MHC molecule, there should be sig- 
nificant structural differences in the receptor from clone to 
clone; each T-cell clone should have an antigenic marker 
similar to the idiotype markers that characterize monoclonal 
antibodies. Using this approach, researchers in the early 
1980s isolated the receptor and found that it was a het- 
erodimer consisting of a and (3 chains. 

When antisera were prepared using a(3 heterodimers iso- 
lated from membranes of various T-cell clones, some antis- 
era bound to ap heterodimers from all the clones, whereas 
other antisera were clone specific. This finding suggested that 
the amino acid sequences of the TCR a and (3 chains, like 
those of the immunoglobulin heavy and light chains, have 
constant and variable regions. Later, a second type of TCR 
heterodimer consisting of 8 and 7 chains was identified. In 
human and mouse, the majority of T cells express the ap het- 
erodimer; the remaining T cells express the 78 heterodimer. 
As described below, the exact proportion of T cells expressing 
a(3 or 78 TCRs differs by organ and species, but a(3 T cells 
normally predominate. 

The TCR P-Chain Gene Was Cloned by Use 
of Subtractive Hybridization 

In order to identify and isolate the TCR genes, S. M. Hedrick 
and M. M. Davis sought to isolate mRNA that encodes the a 
and (3 chains from a T H -cell clone. This was no easy task be- 
cause the receptor mRNA represents only a minor fraction of 
the total cell mRNA. By contrast, in the plasma cell, im- 
munoglobulin is a major secreted cell product, and mRNAs 
encoding the heavy and light chains are abundant and easy to 
purify. 

The successful scheme of Hedrick and Davis assumed that 
the TCR mRNA— like the mRNAs that encode other integral 
membrane proteins — would be associated with membrane- 
bound polyribosomes rather than with free cytoplasmic ri- 
bosomes. They therefore isolated the membrane-bound 
polyribosomal mRNA from a T H -cell clone and used reverse 
transcriptase to synthesize 32 P-labeled cDNA probes (Figure 
9-2). Because only 3% of lymphocyte mRNA is in the 
membrane-bound polyribosomal fraction, this step elimi- 
nated 97% of the cell mRNA. 

Hedrick and Davis next used a technique called DNA sub- 
tractive hybridization to remove from their preparation the 
[ 32 P] cDNA that was not unique to T cells. Their rationale for 
this step was based on earlier measurements by Davis show- 
ing that 98% of the genes expressed in lymphocytes are com- 
mon to B cells and T cells. The 2% of the expressed genes that 



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853 6d_ch0 9_2 0-22 



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' Immunology 5e: 



ofB-CellandT-Cell Respons 




mRNA 

97% in free 3% in 

lie membrane-bound 

polyribosomes polyribosomes 





is unique to T cells should include the genes encoding the T- 
cell receptor. Therefore, by hybridizing B-cell mRNA with 
their T H -cell [ 32 P]cDNA, they were able to remove, or sub- 
tract, all the cDNA that was common to B cells and T cells. 
The unhybridized [ 32 P]cDNA remaining after this step pre- 
sumably represented the expressed polyribosomal mRNA 
that was unique to the T H -cell clone, including the mRNA 
encoding its T-cell receptor. 

Cloning of the unhybridized [ 32 P]cDNA generated a li- 
brary from which 10 different cDNA clones were identified. 
To determine which of these T-cell-specific cDNA clones 



■ Production and identification of a cDNA clone en- 
coding the T-cell receptor. The flow chart outlines the procedure used 
by S. Hedrick and M. Davis to obtain [ 32 P]cDNA clones correspond- 
ing to T-cell-specific mRNAs. The technique of DNA subtractive hy- 
bridization enabled them to isolate [ 32 P]cDNA unique to the T cell. 
The labeled T H -cell cDNA clones were used as probes (inset) in 
Southern-blot analyses of genomic DNA from liver cells, B-lym- 
phoma cells, and six different T H -cell clones (a-f). Probing with 
cDNA clone 1 produced a distinct blot pattern for each T-cell clone, 



whereas probing with cDNA c 


lone 2 did not. Assuming that liver 


cells and B cells contained un 


earranged germ-line TCR DNA, and 


that each of the T-cell clones 


contained different rearranged TCR 


genes, the results using cDNA 


clone 1 as the probe identified clone 


1 as the T-cell-receptor gene. 


The cDNA of clone 2 identified the 


gene for another T-cell memb 


ane molecule encoded by DNA that 


does not undergo rearrangem 


nt. [Based on S. Hedrick et al., 1984, 


Nature 308:149.] 





represented the T-cell receptor, all were used as probes to 
look for genes that rearranged in mature T cells. This ap- 
proach was based on the assumption that, since the a(3 T-cell 
receptor appeared to have constant and variable regions, its 
genes should undergo DNA rearrangements like those ob- 
served in the Ig genes of B cells. The two investigators tested 
DNA from T cells, B cells, liver cells, and macrophages by 
Southern-blot analysis using the 10 [ 32 P]cDNA probes to 
identify unique T-cell genomic DNA sequences. One clone 
showed bands indicating DNA rearrangement in T cells but 
not in the other cell types. This cDNA probe identified six 
different patterns for the DNA from six different mature T- 
cell lines (see Figure 9-2 inset, upper panel). These different 
patterns presumably represented rearranged TCR genes. 
Such results would be expected if rearranged TCR genes oc- 
cur only in mature T cells. The observation that each of the 
six T-cell lines showed different Southern-blot patterns was 
consistent with the predicted differences in TCR specificity 
in each T-cell line. 

The cDNA clone 1 identified by the Southern-blot analy- 
ses shown in Figure 9-2 has all the hallmarks of a putative 
TCR gene: it represents a gene sequence that rearranges, is 
expressed as a membrane-bound protein, and is expressed 
only in T cells. This cDNA clone was found to encode the (3 
chain of the T-cell receptor. Later, cDNA clones were identi- 
fied encoding the a chain, the 7 chain, and finally the 8 chain. 
These findings opened the way to understanding the T-cell 
receptor and made possible subsequent structural and func- 
tional studies. 



ap and 70 T-Cell Receptors: 
Structure and Roles 

The domain structures of a (3 and 78 TCR heterodimers 
are strikingly similar to that of the immunoglobulins; 



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' Immunology 5e: 



thus, they are classified as members of the immunoglobulin 
superfamily (see Figure 4-19). Each chain in a TCR has two 
domains containing an intrachain disulfide bond that spans 
60-75 amino acids. The amino-terminal domain in both 
chains exhibits marked sequence variation, but the sequences 
of the remainder of each chain are conserved. Thus the TCR 
domains-one variable (V) and one constant (C)-are struc- 
turally homologous to the V and C domains of immuno- 
globulins, and the TCR molecule resembles an Fab fragment 
(Figure 9-3). The TCR variable domains have three hyper- 
variable regions, which appear to be equivalent to the 
complementarity determining regions (CDRs) in immuno- 
globulin light and heavy chains. There is an additional area of 
hypervariability (HV4) in the (3 chain that does not normally 
contact antigen and therefore is not considered a CDR. 

In addition to the constant domain, each TCR chain con- 
tains a short connecting sequence, in which a cysteine residue 
forms a disulfide link with the other chain of the het- 
erodimer. Following the connecting region is a transmem- 
brane region of 21 or 22 amino acids, which anchors each 
chain in the plasma membrane. The transmembrane do- 
mains of both chains are unusual in that they contain posi- 
tively charged amino acid residues. These residues enable the 
chains of the TCR heterodimer to interact with chains of the 
signal-transducing CD3 complex. Finally, each TCR chain 



i short cytoplasmic tail of 5-12 amino acids at the 
carboxyl-terminal end. 

a(3 and 78 T-cell receptors were initially difficult to inves- 
tigate because, like all transmembrane proteins, they are in- 
soluble. This problem was circumvented by expressing 
modified forms of the protein in vitro that had been engi- 
neered to contain premature in-frame stop codons that pre- 
clude translation of the membrane-binding sequence that 
makes the molecule insoluble. 

The majority of T cells in the human and the mouse ex- 
press T-cell receptors encoded by the a(3 genes. These recep- 
tors interact with peptide antigens processed and presented 
on the surface of antigen-presenting cells. Early indications 
that certain T cells reacted with nonpeptide antigens were 
puzzling until some light was shed on the problem when 
products of the CD1 family of genes were found to present 
carbohydrates and lipids. More recently, it has been found 
that certain 78 cells react with antigen that is neither 
processed nor presented in the context of a MHC molecules. 

Differences in the antigen-binding regions of a(i and 78 
were expected because of the different antigens they recog- 
nize, but no extreme dissimilarities were expected. However, 
the recently completed three-dimensional structure for a 78 
receptor that reacts with a phosphoantigen, reported by 
Allison, Garboczi, and their coworkers, reveals significant 




Sell- 



ing the: 



■bound IgM or 
nains with the i 



ity between the a(3 T-cell receptor and n 

cells. The TCR a and (3 chain each contair 

munoglobulin-fold structure. The amino-terminal domains (V„ a 

Vp) exhibit sequence variation and contain three hypervariable 

gions equivalent to the CDRs in antibodies. The sequence of the a 

stant domains (C„ and C p ) does not vary. The two TCR chains ; 

connected by a disulfide bond between their constant sequences; 1 



IgM H chains are connected to one another by a disulfide bond in the 
hinge region of the H chain, and the L chains are connected to the H 
chains by disulfide links between the C termini of the L chains and 
the C^ region. TCR molecules interact with CD3 via positively 
charged amino acid residues (indicated by +) in their transmem- 
brane regions. Numbers indicate the length of the chains in the TCR 
molecule. Unlike the antibody molecule, which is bivalent, the TCR is 
monovalent. 



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ofB-CellandT-Cell Respons 



differences in the overall structures of the two receptor types, 
pointing to possible functional variation. The receptor they 
studied was composed of the 79 and 82 chains, which are 
those most frequently expressed in human peripheral blood. 
A deep cleft on the surface of the molecule accommodates 
the microbial phospholipid for which the 78 receptor is spe- 
cific. This antigen is recognized without MHC presentation. 

The most striking feature of the structure is how it differs 
from the a (3 receptor in the orientation of its V and C re- 
gions. The so-called elbow angle between the long axes of the 
V and C regions of 78 TCR is 1 1 1°; in the a (3 TCR, the elbow 
angle is 149°, giving the molecules distinct shapes (Figure 
9-4). The full significance of this difference is not known, 
but it could contribute to differences in signaling mecha- 
nisms and in how the molecules interact with coreceptor 
molecules. 

The number of 78 cells in circulation is small compared 
with cells that have a(3 receptors, and the V gene segments of 
78 receptors exhibit limited diversity. As seen from the data 
in Table 9-1, the majority of 78 cells are negative for both 
CD4 and CD8, and most express a single 78-chain subtype. 
In humans the predominant receptor expressed on circulat- 
ing 78 cells recognizes a microbial phospholipid antigen, 3- 
formyl-1 -butyl pyrophosphate, found on M. tuberculosis and 
other bacteria and parasites. This specificity for frequently 
encountered pathogens led to speculation that 78 cells may 
function as an arm of the innate immune response, allowing 
rapid reactivity to certain antigens without the need for a 
processing step. Interestingly, the specificity of circulating 78 
cells in the mouse and of other species studied does not par- 
allel that of humans, suggesting that the 78 response may be 
directed against pathogens commonly encountered by a 
given species. Furthermore, data indicating that 78 cells can 
secrete a spectrum of cytokines suggest that they may play a 
regulatory role in recruiting a (3 T cells to the site of invasion 
by pathogens. The recruited a|3 T cells would presumably 
display a broad spectrum of receptors; those with the highest 




Feature 


a(3T cells 


78 T cells 


Proportion of CD3 + 
cells 


90-99% 


1-10% 


TCR V gene germ- 


Large 


Small 


CD4/CD8 
phenotype 






CD4 + 


-60% 


<1% 


CD8 + 


-30% 


-30% 


CD4 + CD8 + 


<1% 


<1% 


CD4~CD8~ 


<1% 


-60% 


MHC restriction 


CD4 + : MHC 
CD8 + : MHC 


No MHC 


Ligands 


Peptide + MHC 


Phospholipid 


SOURCE: D. Kabelitz et a 
21:55, p. 36. 


, 1999, Springer Seminars in 1 


nmunopatholog, 



• Comparison of the 78 TCR and ap TCR. The dif- 
ference in the elbow angle is highlighted with black lines. [From 
T. Allison et ai, 2001, Nature 47 7: 820.] 



affinity would be selectively activated and amplified to deal 
with the pathogen. 



Organization and Rearrangement 
of TCR Genes 

The genes that encode the a (3 and 78 T-cell receptors are ex- 
pressed only in cells of the T-cell lineage. The four TCR loci 
(a, (3, 7, and 8) are organized in the germ line in a manner 
that is remarkably similar to the multigene organization of 
the immunoglobulin (Ig) genes (Figure 9-5). As in the case 
of Ig genes, functional TCR genes are produced by re- 
arrangements of V and J segments in the ct-chain and 7- 
chain families and V, D, and J segments in the (3-chain and 
8-chain families. In the mouse, the a-, (3-, and 7-chain gene 
segments are located on chromosomes 14, 6, and 13, respec- 
tively. The 8-gene segments are located on chromosome 14 
between the V a and J a segments. The location of the 8-chain 
gene family is significant: a productive rearrangement of the 
a-chain gene segments deletes Cg, so that, in a given T cell, 
the a{3 TCR receptor cannot be coexpressed with the 78 

Mouse germ-line DNA contains about 100 V a and 50 J a 
gene segments and a single C a segment. The 8-chain gene 
family contains about 10 V gene segments, which are largely 
distinct from the V a gene segments, although some sharing 



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49 AM Page 205 n 



' Immunology 5e: 



Mouse TCR a < uomosome 14) 

(V a « = -100 ; V 6 « = -10) Q a n = -50) 

LV a l LV a 2 LV a n L Vgl L Vg« D 5 1D 8 2J 5 1J 6 2 C 8 L V 6 5 J H 1 J a 2 J a 3 J a « C a 

■ICbHHHH]-E3ICHHH] D-EIhi-* 



Mouse TCR p-chain DNA (chromosome 6) 

(VjjM = 20 - 30) 

LVpl LV p 2 LV p « Dpi Jpl.l-Jpl.7 Cpl D p 2 Jp2.1-J p 2.7 C p 2 LV p 14 

MniiM r« i< /■' i ■■ 1 1 ■ una (i iii-ni ■ i ■ ■ 

L V y 5 L V y 2 L V y 4 L V y 3 J y l C y l L J y 3 C y 3 C y 2 J y 2 L L J y 4 C y 4 



ine organization of the mouse TCR a-, £-, y-, the various gene segments differs in some cases (see Table 9-2). 

nents. Each C gene segment is composed of a {Adapted from D. Raulet, 1989, Annu. Rev. Immunol. 7:175, and M. 

trons, which are not shown. The organization Davis, 1990, Annu. Rev. Biochem. 59:475.] 
5 in humans is similar, although the number of 



of V segments has been observed in rearranged a- and 
8-chain genes. Two D s and two Jg gene segments and one C§ 
segment have also been identified. The p-chain gene family 
has 20-30 V gene segments and two almost identical repeats 
of D, J, and C segments, each repeat consisting of one Dp, six 
Jp, and one Cp. The 7-chain gene family consists of seven V 7 
segments and three different functional ] y -C y repeats. The 
organization of the TCR multigene families in humans is 
generally similar to that in mice, although the number of seg- 
ments differs (Table 9-2). 



| TABLE 9-2 [ 




Chromosome 
location 




. OF GE 


vJ E SEGN 


ENTS 


Gene 


V 


D 


J 


C 


a Chain 


14 


50 




70 


1 


5 Chain* 


14 


3 


3 


3 


1 


p Chain 1 " 


7 


57 


2 


13 


2 


y Chain* 


7 


14 




5 


2 



ining2or3J 1 and1 C,. 



TCR Variable-Region Genes Rearrange 
in a Manner Similar to Antibody Genes 

The a chain, like the immunoglobulin L chain, is encoded by 
V, J, and C gene segments. The (3 chain, like the im- 
munoglobulin H chain, is encoded by V, D, J, and C gene seg- 
ments. Rearrangement of the TCR a- and (3-chain gene 
segments results in VJ joining for the a chain and VDJ join- 
ing for the f3 chain (Figure 9-6). 

After transcription of the rearranged TCR genes, RNA 
processing, and translation, the a and p chains are expressed 
as a disulfide-linked heterodimer on the membrane of the T 
cell. Unlike immunoglobulins, which can be membrane 
bound or secreted, the a(3 heterodimer is expressed only in a 
membrane-bound form; thus, no differential RNA process- 
ing is required to produce membrane and secreted forms. 
Each TCR constant region includes a connecting sequence, a 
transmembrane sequence, and a cytoplasmic sequence. 

The germ-line DNA encoding the TCR a and |3 chain 
constant regions is much simpler than the immunoglobulin 
heavy-chain germ-line DNA, which has multiple C gene seg- 
ments encoding distinct isotypes with different effector func- 
tions. TCR a-chain DNA has only a single C gene segment; 
the p-chain DNA has two C gene segments, but their protein 
products differ by only a few amino acids and have no known 
functional differences. 

MECHANISM OF TCR DNA REARRANGEMENTS 
The mechanisms by which TCR germ-line DNA is re- 
arranged to form functional receptor genes appear to be 



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ofB-CeilandT-Cell Respons 



VISUALIZING CONCEPTS 



LV a l LV a « LV 8 1 LV 8 « D 6 1D S 2 J 6 1J 5 2 C 5 L V 6 5 J a lJ„2J„M C K 

Germ-linea-chainDNA 5'_| a .... | £^|£] |£^HWH}^^ICHHM- ' ' " C3" 3' 



Rearranged a-chain DNA 



Protein product rap heterodimer 



Rearranged |3- chain DNA 



Germ-line |3-chain DNA 5-1 





LVpDpJp Cp Dp2 Jp Cp2 LVpl4 



Jp Cpl Dp2 Jp C p 2 LVpl4 



tional gene encoding the a{3 T-cell receptor. The a-chain DNA, 
analogous to immunoglobulin light-chain DNA, undergoes a 
variable-region V Q -J„ joining. The (3-chain DNA, analogous to im- 
munoglobulin heavy-chain DNA, undergoes two variable-region 
joinings: first Dp to J p and then V p to D p J p . Transcription ofthe re- 
arranged genes yields primary transcripts, which are processed to 
give mRNAs encoding the a and p chains ofthe membrane- 



bound TCR. The leader sequence is cleaved from the nascent 
polypeptide chain and is not present in the finished protein. As 
no secreted TCR is produced, differential processing ofthe pri- 
mary transcripts does not occur. Although the (3-chain DNA con- 
tains two C genes, the gene products of these two C genes exhibit 
no known functional differences. The C genes are composed of 
several exons and introns, which are not individually shown here 
(see Figure 9-7). 



similar to the mechanisms of Ig-gene rearrangements. For 
example, conserved heptamer and nonamer recombination 
signal sequences (RSSs), containing either 12-bp (one-turn) 
or 23-bp (two-turn) spacer sequences, have been identified 
flanking each V, D, and J gene segment in TCR germ-line 
DNA (see Figure 5-6). All of the TCR-gene rearrangements 
follow the one-turn/two-turn joining rule observed for the Ig 
genes, so recombination can occur only between the two dif- 
ferent types of RSSs. 

Like the pre-B cell, the pre-T cell expresses the recombi- 
nation-activating genes (RAG-1 and RAG-2). The RAG- 1/2 
recombinase enzyme recognizes the heptamer and non- 
amer recognition signals and catalyzes V-J and V-D-J join- 
ing during TCR-gene rearrangement by the same deletional 
or inversional mechanisms that occur in the Ig genes 
(see Figure 5-7). As described in Chapter 5 for the 



immunoglobulin genes, RAG- 1/2 introduces a nick on one 
DNA strand between the coding and signal sequences. The 
recombinase then catalyzes a transesterification reaction 
that results in the formation of a hairpin at the coding 
sequence and a flush 5' phosphorylated double-strand 
break at the signal sequence. Circular excision products 
thought to be generated by looping-out and deletion dur- 
ing TCR-gene rearrangement have been identified in thy- 
mocytes (see Figure 5-8). 

Studies with SCID mice, which lack functional T and B 
cells, provide evidence for the similarity in the mechanisms 
of Ig-gene and TCR-gene rearrangements. As explained in 
Chapter 19, SCID mice have a defect in a gene required for 
the repair of double-stranded DNA breaks. As a result of this 
defect, D and J gene segments are not joined during re- 
arrangement of either Ig or TCR DNA (see Figure 5-10). This 



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:79 Mac 79:45__BW:C 



finding suggests that the same double-stranded break-repair 
enzymes are involved in V-D-J rearrangements in B cells and 
in T cells. 

Although B cells and T cells use very similar mechanisms 
for variable- region gene rearrangements, the Ig genes are not 
normally rearranged in T cells and the TCR genes are not re- 
arranged in B cells. Presumably, the recombinase enzyme sys- 
tem is regulated in each cell lineage, so that only 
rearrangement of the correct receptor DNA occurs. Re- 
arrangement of the gene segments in both T and B cell cre- 
ates a DNA sequence unique to that cell and its progeny. The 
large number of possible configurations of the rearranged 
genes makes this new sequence a marker that is specific for 
the cell clone. These unique DNA sequences have been used 
to aid in diagnoses and in treatment of lymphoid leukemias 
and lymphomas, cancers that involve clonal proliferation of 
T or B cells (see Clinical Focus on page 208). 

ALLELIC EXCLUSION OF TCR GENES 






This c 



itioned above, the 8 genes a 
complex and are deleted by c 



2 located within the a- 
-chain rearrangements. 



Lt provides an irrevocable mode of exclusion for the 
8 genes located on the same chromosome as the rearranging 
a genes. Allelic exclusion of genes for the TCR a and (3 chains 
occurs as well, but exceptions have been observed. 

The organization of the (3-chain gene segments into two 
clusters means that, if a nonproductive rearrangement oc- 
curs, the thymocyte can attempt a second rearrangement. 
This increases the likelihood of a productive rearrangement 
for the (3 chain. Once a productive rearrangement occurs for 
one (3-chain allele, the rearrangement of the other (3 allele is 
inhibited. 

Exceptions to allelic exclusion are most often seen for the 
TCR a-chain genes. For example, analyses of T-cell clones 
that express a functional a(3 T-cell receptor revealed a num- 
ber of clones with productive rearrangements of both a- 
chain alleles. Furthermore, when an immature T-cell 
lymphoma that expressed a particular a(3 T-cell receptor was 
subcloned, several subclones were obtained that expressed 
the same (3-chain allele but an a-chain allele different from 
the one expressed by the original parent clone. Studies with 
transgenic mice also indicate that allelic exclusion is less 
stringent for TCR a-chain genes than for (3-chain genes. 
Mice that carry a productively rearranged ap-TCR transgene 
do not rearrange and express the endogenous (3-chain genes. 
However, the endogenous a-chain genes sometimes are ex- 
pressed at various levels in place of the already rearranged a- 

Since allelic exclusion is not complete for the TCR a 
chain, there are rare occasions when more than one a chain 
is expressed on the membrane of a given T cell. The obvious 
question is how do the rare T cells that express two a (3 T-cell 
receptors maintain a single antigen-binding specificity? One 
proposal suggests that when a T cell expresses two different 
a(3 T-cell receptors, only one is likely to be self-MHC re- 
stricted and therefore functional. 



Rearranged TCR Genes Are Assembled from 
V, j, and D Gene Segments 

The general structure of rearranged TCR genes is shown in 
Figure 9-7. The variable regions of T-cell receptors are, of 
course, encoded by rearranged VDJ and VJ sequences. In 
TCR genes, combinatorial joining of V gene segments ap- 
pears to generate CDR1 and CDR2, whereas junctional flexi- 
bility and N-region nucleotide addition generate CDR3. 
Rearranged TCR genes also contain a short leader (L) exon 
upstream of the joined VJ or VDJ sequences. The amino acids 
encoded by the leader exon are cleaved as the nascent 
polypeptide enters the endoplasmic reticulum. 

The constant region of each TCR chain is encoded by a C 
gene segment that has multiple exons (see Figure 9-7) corre- 
sponding to the structural domains in the protein (see Figure 
9-3). The first exon in the C gene segment encodes most of 
the C domain of the corresponding chain. Next is a short 
exon that encodes the connecting sequence, followed by ex- 
ons that encode the transmembrane region and the cytoplas- 
mic tail. 

TCR Diversity Is Generated Like Antibody 
Diversity but Without Somatic Mutation 

Although TCR germ-line DNA contains far fewer V gene seg- 
ments than Ig germ-line DNA, several mechanisms that op- 
erate during TCR gene rearrangements contribute to a high 
degree of diversity among T-cell receptors. Table 9-3 (page 
210) and Figure 9-8 (page 211) compare the generation of 
diversity among antibody molecules and TCR molecules. 



CDR1 CDR2 CDR3 ^Z " 

L ^, ^ ^ C a H Tm CT 

Rearranged . _ > ,, _ 

a-chain gene H v I- 1 ! | | U LJ U 



/ 



I Connecting / Cytc 

v Y ' J sequence / plasm 

Encoded Leader Variable Constant Trans- tail 

domains domain domain membrane 

(V a orV p ) (Co.orCp) region 



m™™™ msr-n^^^ 



CDRl CDR2 CDR3 



erratic diagram of rearranged a(3-TCR genes 
showing the exons that encode the various domains of the a(3 T-cell 
receptor and approximate position of the CDRs. Junctional diversity 
(vertical arrows) generates CDR3 (see Figure 9-8). The structures of 
the rearranged 7- and 8-chain genes are similar, although additional 
junctional diversity can occur in 8-chain genes. 



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ofB-CeilandT-Cell Respons 



CLINICAL FOCUS 



T-Cell Rearrangements as 
Markers for Cancerous Cells 



! cancers, which include 
leukemia and lymphoma, involve the un- 
controlled proliferation of a clonal popu- 
lation of T cells. Successful treatment 
requires quick and certain diagnosis in 
order to apply the most effective treat- 
ment. Once treatment is initiated, reli- 
able tests are needed to determine 
whether the treatment regimen was suc- 
cessful. In principle, because T-cell can- 
cers are clonal in nature, the cell 
population that is cancerous could be 
identified and monitored by the expres- 
sion of its unique T-cell receptor mole- 
cules. However, this approach is rarely 
practical because detection of a specific 
TCR molecule requires the tedious and 
lengthy preparation of a specific anti- 
body directed against its variable region 
(an anti-idiotype antibody). Also, surface 
expression of the TCR molecule occurs 
somewhat late in the development of the 
T cell, so cancers stemming from T cells 
that have not progressed beyond an early 
stage of development will not display a 
TCR molecule and will not be detected by 
the antibody. An alternative means of 
identifying a clonal population of T cells 
is to look at their DNA rather than pro- 
tein products. The pattern resultingfrom 
rearrangement of the TCR genes can 
provide a unique marker for the cancer- 
ous T cell. Because rearrangement of 



the TCR genes in the T cells occurs be- 
fore the product molecule is expressed, 
T cells in early stages of development 
can be detected. The unique gene frag- 
ments that result from TCR gene re- 
arrangement can be detected by sim- 
ple molecular-biological techniques and 
provide a true fingerprint for a clonal 
cell population. 

DNA patterns that result from re- 
arrangement of the genes in the TCR (3 
region are used most frequently as mark- 
ers. There are approximately 50 V p gene 
segments that can rearrange to one of 
two D-region gene segments and subse- 
quently to one of 12 J gene segments 
(see Figure 9-8). Because each of the 50 
or so V-region genes is flanked by unique 
sequences, this process creates new 
DNA sequences that are unique to each 
cell that undergoes the rearrangement; 
these new sequences may be detected by 
Southern-blot techniques or by PCR 
(polymerase chain reaction). Since the 
entire sequence of the D, J, and C region 
of the TCR gene (3 complex is known, the 
appropriate probes and restriction en- 
zymes are easily chosen for Southern 
blotting (see diagram). 

Detection of rearranged TCR DNA 
may be used as a diagnostic tool when 
abnormally enlarged lymph nodes per- 
sist; this condition could result either 
from inflammation due to chronic infec- 



tion or from proliferation of a 
lymphoid cell. If inflammation is the 
cause, the cells would come from a vari- 
ety of clones, and the DNA isolated 
from them would be a mixture of many 
different TCR sequences resulting from 
multiple rearrangements; no unique 
fragments would be detected. If the per- 
sistent enlargement of the nodes repre- 
sents a clonal proliferation, there would 
be a detectable DNA fragment, because 
the cancerous cells would all contain 
the same TCR DNA sequence produced 
by DNA rearrangement in the parent 
cell. Thus the question whether the ob- 
served enlargement was due to the can- 
cerous growth of T cells could be 
answered by the presence of a single 
new gene fragment in the DNA from the 
cell population. Because Ig genes re- 
arrange in the same fashion as the TCR 
genes, similartechniques use Ig probes 
to detect clonal B-cell populations by 
their unique DNA patterns. The tech- 
nique, therefore, has value for a wide 
range of lymphoid-cell cancers. 

Although the detection of a unique 
DNA fragment resultingfrom rearranged 
TCR or Ig genes indicates clonal prolifer- 
ation and possible malignancy of T or B 
cells, the absence of such a fragment 
does not rule out cancer of a population 
of lymphoid cells. The cell involved may 
not contain rearranged TCR or Ig genes 
that can be detected by the method used, 
either because of its developmental stage 
or because it is of another lineage (78 T 
cells, for example). 

If the DNA fragment test and other di- 
agnostic criteria indicate that the patient 
has a lymphoid cell cancer, treatment by 



Combinatorial joining of variable-region gene segments 
generates a large number of random gene combinations for 
all the TCR chains, as it does for the Ig heavy- and light- 
chain genes. For example, 100 V a and 50 J a gene segments 
can generate 5 X 10 3 possible VJ combinations for the TCR 
a chain. Similarly, 25 V p , 2 D p , and 12 J p gene segments can 
give 6 X 10 2 possible combinations. Although there are 



fewer TCR V a and V p gene segments than immunoglobulin 
V H and V a segments, this difference is offset by the greater 
number of J segments in TCR germ-line DNA. Assuming 
that the antigen-binding specificity of a given T-cell receptor 
depends upon the variable region in both chains, random 
association of 5 X 10 3 V a combinations with 6 X 10 2 V p 
combinations can generate 3 X 10 6 possible combinations 



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8536d:Goldsby et al . / ] 



EcoRl EcoRl 



EcoRl 4 , ,. h EcoR\ 



Germ-line p-chain DNA 5 - 




Rearranged p-chain DNA 5'- 



Digestion of human TCR (3-chain DNA in a germ-l 
EcoRl and then probing with a C-region sequence \ 

:s by Southern blotting. When the DNA has rearranged, 
excised. Digestion with EcoRl will yield a different fragment unique to the specific V p and J p 
region gene segments incorporated into the rearranged gene, as indicated in this hypothetical 
example. The technique used for this analysis derives from that first used by S. M. Hedrick 
and his coworkers to detect unique TCR |3 genes in a series of mouse T-cell clones (see inset 
to Figure 9-2). For highly sensitive detection of the rearranged TCR sequence, the polymerase 
chain reaction (PCR) is used. The sequence of the 5' primer (red bar) is based on a unique 
sequence in the (V p ) gene segment used by the cancerous clone (Vp2 in this example) and 
the 3' primer (red bar) is a constant-region sequence. For chromosomes on which this V gene 
is not rearranged, the fragment will be absent because it is too large to be efficiently amplified. 



blot probed 
with C DNA 



radiation therapy or chemotherapy would 
follow. The success of this treatment can 
be monitored by probing DNA from the 
patient for the unique sequence found in 
the cancerous cell. If the treatment regi- 
men is successful, the number of cancer- 
ous cells will decline greatly. If the 
number of cancerous cells falls below 1% 
or 2% of the total T-cell population, 
analysis by Southern blot may no longer 
detect the unique fragment. In this case, 
a more sensitive technique, PCR, may be 
used. (With PCR it is possible to am- 



plify, or synthesize multiple copies of, a 
specific DNA sequence in a sample; 
primers can hybridize to the two ends of 
that specific sequence and thus direct a 
DNA polymerase to copy it; see Figure 
23-1 3 for details.) To detect a portion of 
the rearranged TCR DNA, amplification 
using a sequence from the rearranged V 
region as one primer and a sequence 
from the p-chain C region as the other 
primer will yield a rearranged TCR DNA 
fragment of predicted size in sufficient 
quantity to be detected by electrophore- 



sis (see red arrow in the diagram). Re- 
cently, quantitative PCR methods have 
been used to follow patients who are in 
remission in order to make decisions 
about resuming treatment if the num- 
ber of cancerous cells, as estimated by 



thes 



schniq 



tain level. Therefore, the presence of the 
rearranged DNA in the clonal popula- 
tion of T cells gives the clinician a valu- 
able tool for diagnosing lymphoid-cell 
cancer and for monitoring the progress 
of treatment. 



for the a(3 T-cell receptor. Additional means to generate di- 
versity in the TCR V genes are described below, so 3 X 10 6 
combinations represents a minimum estimate. 

As illustrated in Figure 9-8b, the location of one-turn 
(12-bp) and two-turn (23-bp) recombination signal se- 
quences (RSSs) in TCR (3- and 8-chain DNA differs from 



the RSSs in TCR germ-line DNA, alternative joining of D 
gene segments can occur while the one-turn/two-turn join- 
ing rule is observed. Thus, it is possible for a V p gene seg- 
ment to join directly with a J p or a D p gene segment, 
generating a (VJ) p or (VDJ) p unit. 

Alternative joining of h-chain gene segments generates 






g heavy-chain DNA. Because of the arrangement of similar units; in addition, one D 8 can join with another, 



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8536d:Goldsby et al . / ] 



3 f B-Cell and T-Cell Respons 



| TABLE 9-3 [ 




IMMUNOGLOBULINS 


«P T-CELL RECEPTOR 


78 T-CELL RECEPTOR 


Mechanism of diversity 


H Chain k Chain 


a Chain fj Chain 


-y Chain 8 Chain 




ESTIMATED NU 


i/IBER OF SEGMENTS 





Multiple gerr 
segments 



POSSIBLE NUMBER OF COMBINATIONS 



Combinatory 


IV-j 


andV-D-j j 


oining 


Alternative jo 


'""? 


of D gene segments 


Junctional fie 


<ibility 


N-region nuc 


eotide ad 


P-region nuc 


eotideadc 


Somatic mut 


tion 


Combinatory 


1 


association 


of chains 


"A plus sign (+ 


indicates n 


A minus sign (- 





is generated by N-regior 



yielding (VDDJ) 8 and, in humans, (VDDDJ) S . This mecha- 
nism, which cannot operate in Ig heavy-chain DNA, gener- 
ates considerable additional diversity in TCR genes. 

The joining of gene segments during TCR-gene re- 
arrangement exhibits junctional flexibility. As with the Ig 
genes, this flexibility can generate many nonproductive re- 
arrangements, but it also increases diversity by encoding sev- 
eral alternative amino acids at each junction (see Figure 
9-8c). In both Ig and TCR genes, nucleotides may be added at 
the junctions between some gene segments during re- 
arrangement (see Figure 5-15). Variation in endonuclease 
cleavage leads to the addition of further nucleotides that are 
palindromic. Such P-region nucleotide addition can occur 
in the genes encoding all the TCR and Ig chains. Addition of 
N-region nucleotides, catalyzed by a terminal deoxynu- 
cleotidyl transferase, generates additional junctional diver- 
sity. Whereas the addition of N-region nucleotides in 
immunoglobulins occurs only in the Ig heavy-chain genes, it 
occurs in the genes encoding all the TCR chains. As many as 
six nucleotides can be added by this mechanism at each junc- 
tion, generating up to 5461 possible combinations, assuming 



random selection of nucleotides (see Figure 9-8d). Some of 
these combinations, however, lead to nonproductive re- 
arrangements by inserting in-frame stop codons that prema- 
turely terminate the TCR chain, or by substituting amino 
acids that render the product nonfunctional. Although each 
junctional region in a TCR gene encodes only 10-20 amino 
acids, enormous diversity can be generated in these regions. 
Estimates suggest that the combined effects of P- and N- 
region nucleotide addition and joining flexibility can gener- 
ate as many as 10 13 possible amino acid sequences in the TCR 
junctional regions alone. 

The mechanism by which diversity is generated for the 
TCR must allow the receptor to recognize a very large num- 
ber of different processed antigens while restricting its 
MHC-recognition repertoire to a much smaller number of 
self-MHC molecules. TCR DNA has far fewer V gene seg- 
ments than Ig DNA (see Table 9-3). It has been postulated 
that the smaller number of V gene segments in TCR DNA 
have been selected to encode a limited number of CDR1 and 
CDR2 regions with affinity for regions of the a helices of 
MHC molecules. Although this is an attractive idea, it is 



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8536d:Goldsby et al . / ] 



T-Cell Receptor chai 



T-CELL RECEPTOR 

(a) Combinatorial V-J and V-D-J joining 



IMMUNOG-L: 



V DJ 
| | | (3 and 8 chains 




V D.I 
~[J] H chain 


V J 
| | a and y chains 




V J 
J] L chain 


(b) Alternative joining of D gene segments 






L V 5 D 5 D S J S 




L V H D H J H 


i 


A = One-turn 

RSS 


1 


V 5 -D 5 -J 5 , 

Va-Dg-Dg-jg 


/ = Two-turn 

RSS 


v H- D H-JH° nl y 








One-turn RSS D 5 




One-turn RSS D H 


cactgtg| gtggact 


CACTGTG ATGGACT 






GATGCTCC CACAGTG 


T G G C C G | CACAGT G 



(d) N-region nucleotide additi 



t = Addition of 

0-6 nucleotides 



Comparison of mechan 
P-region nucleotide addi 



(5461) 2 = 3.0 x 10 7 

V DDJ 
I I I I I 8chai " 

TTT 
(546l) 3 = 1.6 x 10 11 

for generating diversity Ig ger 



addil 



of the expressed chains ge 
in both TCR and among both TCR and Ig molecule- 



nes. Combinatorial 
additional diversity 



made unlikely by recent data on the structure of the TCR- 
peptide-MHC complex showing contact between peptide 
and CDR1 as well as CDR3. Therefore the TCR residues that 
bind to peptide versus those that bind MHC are not confined 
solely to the highly variable CDR3 region. 

In contrast to the limited diversity of CDR1 and CDR2, 
the CDR3 of the TCR has even greater diversity than that 
seen in immunoglobulins. Diversity in CDR3 is generated by 
junctional diversity in the joining of V, D, and J segments, 



joining of multiple D gene segments, and the introduction 
of P and N nucleotides at the V-D-J and V-J junctions (see 
Figure 9-7). 

Unlike the Ig genes, the TCR genes do not appear to un- 
dergo extensive somatic mutation. That is, the functional 
TCR genes generated by gene rearrangements during T-cell 
maturation in the thymus have the same sequences as those 
found in the mature peripheral T-cell population. The 
: mutation in T cells ensures that T-cell 



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853 6d_ch0 9_2 0-22 



49 AM Page 212 n 



' Immunology 5e: 



ofB-CellandT-Cell Respons 



specificity does not change after thymic selection and there- 
fore reduces the possibility that random mutation might 
generate a self-reactive T cell. Although a few experiments 
have provided evidence for somatic mutation of receptor 
genes in T cells in the germinal center, this appears to be the 
exception and not the rule. 



T-Cell Receptor Complex: TCR-CD3 

As explained in Chapter 4, membrane-bound immunoglob- 
ulin on B cells associates with another membrane protein, 
the Ig-a/Ig-(3 heterodimer, to form the B-cell antigen recep- 
tor (see Figure 4-18). Similarly, the T-cell receptor associates 
with CD3, forming the TCR-CD3 membrane complex. In 
both cases, the accessory molecule participates in signal 
transduction after interaction of a B or T cell with antigen; it 
does not influence interaction with antigen. 

The first evidence suggesting that the T-cell receptor is as- 
sociated with another membrane molecule came from ex- 
periments in which fluorescent antibody to the receptor was 
shown to cause aggregation of another membrane protein 
designated CD3. Later experiments by J. P. Allison and 



L. Lanier using cross-linking reagents demonstrated that the 
two chains must be within 12 A. Subsequent experiments 
demonstrated not only that CD3 is closely associated with 
the ap heterodimer but also that its expression is required for 
membrane expression of a(3 and 78 T-cell receptors — each 
heterodimer forms a complex with CD3 on the T-cell mem- 
brane. Loss of the genes encoding either CD3 or the TCR 
chains results in loss of the entire molecular complex from 



CD3is 



a complex of five 
3 form three dimer: 
epsilon chains (ye.), a heterodi 
(8e), and a homodimer of two 



polypeptide chains that 
heterodimer of gamma and 
r of delta and epsilon chains 
chains (££) or a heterodimer 



of zeta and eta chains (£t|) (Figure 9-9). The £ and T| chains are 
encoded by the same gene, but differ in their carboxyl-terminal 
ends because of differences in RNA splicing of the primary 
transcript. About 90% of the CD3 complexes examined to date 
incorporate the (££) homodimer; the remainder have the (£t|) 
heterodimer. The T-cell receptor complex can thus be envi- 
sioned as four dimers: the ct(3 or 78 TCR heterodimer deter- 
mines the ligand-binding specificity, whereas the CD3 dimers 
(ye, 8e, and ££ or £iq) are required for membrane expression of 
the T-cell receptor and for signal transduction. 




COOH COOH 



* Schematic diagram of the TCR-CD3 complex, which 
constitutes the T-cell antigen-binding receptor. The CD3 complex 
consists of the ££ homodimer (alternately, a t,r\ heterodimer) plus 76 
and §€ heterodimers. The external domains of the 7, 8, and € chains 
of CD3 are similar to the immunoglobulin fold, which facilitates their 
with the T-cell receptor and each other. Ic 



also may occur between the oppositely charged tran 
gions in the TCR and CD3 chains. The long cytoplas 
CD3 chains contain a common sequence, the in 
tyrosine-based activation motif (ITAM), which func 
transduction. 



lie tails of the 
nunoreceptor 



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8536d_ch09_200-220 8/22/02 2:51 PM Page 213 n 



8536d:Goldsby et al . / ] 



The 7, 8, and e chains of CD 3 are members of the 
globulin superfamily, each containing an immunoglobulin- 
like extracellular domain followed by a transmembrane 
region and a cytoplasmic domain of more than 40 amino 
acids. The £ chain has a distinctly different structure, with a 
very short external region of only 9 amino acids, a trans- 
membrane region, and a long cytoplasmic tail containing 113 
amino acids. The transmembrane region of all the CD3 
polypeptide chains contains a negatively charged aspartic 
acid residue that interacts with one or two positively charged 
amino acids in the transmembrane region of each TCR 
chain. 

The cytoplasmic tails of the CD 3 chains contain a motif 
called the immunoreceptor tyrosine-based activation mo- 
tif (ITAM). ITAMs are found in a number of other receptors, 
including the Ig-a/Ig-(3 heterodimer of the B-cell receptor 
complex and the Fc receptors for IgE and IgG. The ITAM 
sites have been shown to interact with tyrosine kinases and to 
play an important role in signal transduction. In CD3, the y, 
8, and e chains each contain a single copy of ITAM, whereas 
the I and T) chains contain three copies (see Figure 9-9). The 
function of CD3 in signal transduction is described more 
fully in Chapter 10. 



T-Cell Accessory Membrane 
Molecules 

Although recognition of antigen-MHC complexes is medi- 
ated solely by the TCR-CD3 complex, various other mem- 
brane molecules play important accessory roles in antigen 
recognition and T-cell activation (Table 9-4). Some of these 
molecules strengthen the interaction between T cells and 



antigen-presenting cells or target cells, some act in signal 
transduction, and some do both. 

CD4 and CD8 Coreceptors Bind 
to Conserved Regions of MHC 
Class II or I Molecules. 

T cells can be subdivided into two populations according to 
their expression of CD4 or CD8 membrane molecules. As de- 
scribed in preceding chapters, CD4 + T cells recognize anti- 
gen that is combined with class II MHC molecules and 
function largely as helper cells, whereas CD8 + T cells recog- 
nize antigen that is combined with class I MHC molecules 
and function largely as cytotoxic cells. CD4 is a 55-kDa 
monomeric membrane glycoprotein that contains four ex- 
tracellular immunoglobulin-like domains (Dj-D^, a hy- 
drophobic transmembrane region, and a long cytoplasmic 
tail (Figure 9-10) containing three serine residues that can be 
phosphorylated. CD8 generally takes the form of a disulfide- 
linked a (3 heterodimer or of an act homodimer. Both the 
a and (3 chains of CD8 are small glycoproteins of ap- 
proximately 30-38 kDa. Each chain consists of a single ex- 
tracellular immunoglobulin-like domain, a hydrophobic 
transmembrane region, and a cytoplasmic tail (Figure 9-10) 
containing 25-27 residues, several of which can be phos- 
phorylated. 

CD4 and CD8 are classified as coreceptors based on their 
abilities to recognize the peptide-MHC complex and their 
roles in signal transduction. The extracellular domains of 
CD4 and CD8 bind to the conserved regions of MHC mole- 
cules on antigen-presenting cells (APCs) or target cells. Crys- 
tallographic studies of a complex composed of the class I 
MHC molecule HLA-A2, an antigenic peptide, and a CD8 
aa homodimer indicate that CD8 binds to class I molecules 



FUNCTION 



Member of 
Ig superfamily 



CD4 




Class II MHC 


CD8 




Class 1 MHC 


CD2 (LFA-2) 




CD58 (LFA-3) 


LFA-1 (CD11 


a/CD18) 


ICAM-1 (CD54) 


CD28 




B7 


CTLA-4 




B7 


CD45R 




CD22 


CD5 




CD72 



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853 6d_ch0 9_2 0-22 



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8536d:Goldsby et al . / Immunology 5e-: 



ofB-CeilandT-Cell Respons 




General structure of the CD4 and CD8 coreceptor 
the form of an a(3 heterodimer, or an aa homodime 
CD4 molecule contains four Ig-fold domains; eac 
CD8 molecule contains one. 



by contacting the MHC class I a2 and a3 domains as well as 
having some contact with (^-microglobulin (Figure 9-1 la). 
The orientation of the class I a3 domain changes slightly 
upon binding to CD 8. This structure is consistent with a sin- 
gle MHC molecule binding to CD8; no evidence for the pos- 
sibility of multimeric class I-CD8 complexes was observed. 
Similar structural data document the mode by which CD4 
binds to the class II molecule. The contact between CD4 and 
MHC II involves contact of the membrane-distal domain of 
CD4 with a hydrophobic pocket formed by residues from the 
a2 and (32 domains of MHC II. CD4 facilitates signal trans- 
duction and T-cell activation of cells recognizing class II- 
peptide complexes (Figure 9-1 lb). 

Whether there are differences between the roles played by 
the CD4 and CD8 coreceptors remains open to speculation. 
Despite the similarities in structure, recall that the nature of 
the binding of peptide to class I and class II molecules differs 
in that class I has a closed groove that binds a short peptide 
with a higher degree of specificity. Recent data shown below 
indicate that the angle at which the TCR approaches the pep- 
tide MHC complex differs between class I and II. The differ- 
ences in roles played by the CD4 and CD8 coreceptors may 
be due to these differences in binding requirements. As will 
be explained in Chapter 10, binding of the CD4 and CD8 
molecules serves to transmit stimulatory signals to the T 
cells; the signal-transduction properties of both CD4 and 




s of coreceptor 
ecules. (a) Ribbon diagram showing threi 
an HLA-A2 MHC class I molecule bound 
The HLA-A2 heavy chain is shown in greer 
the CD8 al in red, the CD8 a2 in blue, 
white. A flexible loop ofthe a3 domain (re 



with TCR and MHC [Tri- 
dimensional structure of 
o a CD8 aa homodimer. 
p 2 -microglobuliningold, 
nd the bound peptide in 
dues 223-229) is in con- 



tact with the two CD8 subunits. In this model, the right side of CD8 
would be anchored in the T-cell membrane, and the lower left end of 
the class I MHC molecule (the a3 domain) is attached to the surface 
ofthe target cell, (b) Interaction of CD4 with the class II MHC pep- 
tide complex (pMHCII). [Part (a) from Cao et al., 1997, Nature, 
387:630; part (b) from Wang et al., 2001, PNAS, 98(79): 10799.] 



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8536d:Goldsby et al . / ] 



CD8 are mediated through their cytoplasmic domains. Re- 
cent data on the interaction between CD4 and the peptide - 
class II complex indicates that there is very weak affinity 
between them, suggesting that recruitment of molecules 
involved in signal transduction may be the major role for 
CD4. 

Affinity ofTCR for Peptide-MHC Complexes 
Is Weak Compared with Antibody Binding 

The affinity of T-cell receptors for peptide-MHC complexes 
is low to moderate, with K d values ranging from 10~ 4 
to 10~ 7 M. This level of affinity is weak compared with 



antigen-antibody interactions, which generally have K d val- 
ues ranging from 10~ 6 to 10~ 10 M (Figure 9- 12a). However, 
T-cell interactions do not depend solely on binding by the 
TCR; cell-adhesion molecules strengthen the bond between a 
T cell and an antigen-presenting cell or a target cell. Several 
accessory membrane molecules, including CD2, LFA-1, 
CD28, and CD45R bind independently to other ligands on 
antigen-presenting cells or target cells (see Table 9-4 and 
Figure 9-12b). Once cell-to-cell contact has been made by 
the adhesion molecules, the T-cell receptor may scan the 
membrane for peptide-MHC complexes. During activa- 
tion of a T cell by a particular peptide-MHC complex 



the 









T-cell receptors 



Adhesion Molecules 



Growth Factor Receptor 








' Role of coreceptors in TCR binding affinity, (a) 
Affinity constants for various biologic systems, (b) Schematic dia- 
gram of the interactions between the T-cell receptor and the pep- 
tide-MHC complex and of various accessory molecules with their 
ligands on an antigen-presenting cell (left) or target cell (right). 



Binding of the coreceptors CD4 and CD8 and the other accessory 
molecules to their ligands strengthens the bond between the inter- 
acting cells and/or facilitates the signal transduction that leads to 
activation of the T cell. 



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Generation of B-Cell and T-Cell Respons 




s for the TCR-MHC- 
peptide complex, (a) Model showing the interaction between the 
human TCR (top, yellow) and the HLA-A2 class I MHC molecule 
(bottom, blue) with bound HTLV-I Tax peptide (white and red), (b) 
Backbone tube diagram of the ternary complex of mouse TCR 
bound to the class I MHC H-2K b molecule and peptide (green tube 
numbered P1-P8). CDR1 and 2 of the TCR a-chain variable 
domain (V») are colored pink; CDR 1 and 2 of the |3-chain variable 
domain (V p ) are blue, and the CDR3s of both chains 
of the (3 chain is orange, (c) MHC molecul 



above (i.e., from top of part (a), with the hyper 



of the human TCR a (red) and (3 (yellow) variable chains super- 
imposed on the Tax peptide (white) and the al and a.2 domains 
of the HLA-A2 MHC class I molecule (blue), (d) CDR regions of 
mouse TCR a and (3 chains viewed from above, showing the sur- 
face that is involved in binding the MHC-peptide complex. The 
CDRs are labeled according to their origin (for example, al is 
variable CDR1 from the a chain). HV4 is the fourth hypervariable region of 

variable the (3 chain. [Parts (a) and (c) from D. N. Garboczi et al., 1996, 

en. The Nature 384:134-141, courtesy of D. C. Wiley, Harvard University; 

id from parts (b) and (d) from C. Garcia et al., 1996, Science 274:209, cour- 






>ops (1-4) tesy ofC. Garcia, Scripps Research Institute.] 



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8536d_ch09_200-220 8/22/02 2:51 PM Page 217 



8536d:Goldsby et al . / ] 



cell-adhesion molecules, causing closer contact between 
the interacting cells, which allows cytokines or cytotoxic 
substances to be transferred more effectively. Soon after ac- 
tivation, the degree of adhesion declines and the T cell de- 
taches from the antigen-presenting cell or target cell. Like 
CD4 and CD8, some of these other molecules also function 
as signal-transducers. Their important role is demonstrated 
by the ability of monoclonal antibodies specific for the 
binding sites of the cell-adhesion molecules to block T-cell 
activation. 



Three-Dimensional Structures 
ofTCR-Peptide-MHC Complexes 

The interaction between the T-cell receptor and an antigen 
bound to an MHC molecule is central to both humoral and 
cell-mediated responses. The molecular elements of th: 
teraction have now been described in detail by x-ray crys- 
tallography for TCR molecules binding to peptide-MHC 
class I and class II complexes. A three-dimensional struc- 
ture has been determined for the trimolecular comple: 
eluding TCR a and (3 chains and an HLA-A2 molecu 
which an antigenic peptide is bound. Separate studie 
scribe a mouse TCR molecule bound to peptides com- 
plexed with the mouse class I molecule H-2K b and with the 
mouse class II IA* molecule. The comparisons of the TCR 
complexed with either class I or class II suggest that there 
are differences in how the TCR contacts the MHC-peptide 
complex. Newly added to our library of TCR structures is 
that of a 78 receptor bound to an antigen that does not re- 
quire processing. 

From x-ray analysis, the TCR-peptide-MHC complex 
consists of a single TCR molecule bound to a single MHC 
molecule and its peptide. The TCR contacts the MHC mole- 
cule through the TCR variable domains (Figure 9-13 a,b). 
Although the structures of the constant region of the TCR a 
chain and the MHC a3 domain were not clearly established 
by studies of the crystallized human complexes (see Figure 
9-13a), the overall area of contact and the structure of the 
complete TCR variable regions were clear. The constant re- 
gions were established by studies of the mouse complex, 
which showed the orientation proposed for the human 
models (see Figure 9- 13b). Viewing the MHC molecule with 
its bound peptide from above, we can see that the TCR is sit- 
uated across it diagonally, relative to the long dimension of 
the peptide (Figure 9- 13c). The CDR3 loops of the TCR a 
and (3 chains meet in the center of the peptide; and the 
CDR1 loop of the TCR a chain is at the N terminus of the 
peptide, while CDR1 of the |3 chain is at the C terminus of 
the peptide. The CDR2 loops are in contact with the MHC 
molecule; CDR2a is over the a2 domain alpha helix and 
CDR2|3 over the al domain alpha helix (Figure 9-13c). A 
space-filling model of the binding site viewed from above 
(looking down into the MHC cleft) indicates that the pep- 
tide is buried beneath the TCR and therefore is not seen 



from this angle (Figure 9-13d). The data also show that the 
fourth hypervariable regions of the a and (3 chains are not in 
contact with the antigenic peptide. 

As predicted from data for immunoglobulins, the recog- 
nition of the peptide-MHC complex occurs through the 
variable loops in the TCR structure. CDR1 and CDR3 from 
both the TCR a and the TCR |3 chain contact the peptide 
and a large area of the MHC molecule. The peptide is 
buried (see Figure 9- 13d) more deeply in the MHC mole- 
cule than it is in the TCR, and the TCR molecule fits across 
the MHC molecule, contacting it through a flat surface of 
the TCR at the "high points" on the MHC molecule. The 
fact that the CDR1 region contacts both peptide and MHC 
suggests that regions other than CDR3 are involved in pep- 
tide binding. 

TCRs Interact Differently with Class I 
and Class II Molecules 

Can the conclusions drawn from the three-dimensional 
structure of TCR-peptide-class I complexes be extrapo- 
lated to interactions of TCR with class II complexes? Ellis 
Reinherz and his colleagues resolved this question by analy- 
sis of a TCR molecule in complex with a mouse class II mol- 
ecule and its specific antigen. While the structures of the 
peptide-binding regions in class I and class II molecules are 
similar, Chapter 7 showed that there are differences in how 
they accommodate bound peptide (see Figures 7-10a and 
b). A comparison of the interactions of a TCR with class I 
MHC-peptide and class II-peptide reveals a significant 
difference in the angle at which the TCR molecule sits on 
the MHC complexes (Figure 9-14). Also notable is a greater 
number of contact residues between TCR and class II 
MHC, which is consistent with the known higher affinity of 
interaction. However, it remains to be seen whether the ev- 
ident difference in the number of contact points will be true 
for all class I and II structures. 




II MHC 



Comparison of the interactions between a(3 TCR 
and (a) class I MHC-peptide, and (b) class II MHC-peptide. The 
TCR (wire diagram) is red in (a), blue-green in (b); the MHC mole- 
cules are shown as surface models; peptide is shown as ball and 
stick. [From Reinherz et al., 1999, Science 286:1913.] 



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ofB-CeilandT-Cell Respons 



Alloreactivity of T Cells 

The preceding sections have focused on the role of MHC 
molecules in the presentation of antigen to T cells and the in- 
teractions of TCRs with peptide-MHC complexes. However, 
as noted in Chapter 7, MHC molecules were first identified 
because of their role in rejection of foreign tissue. Graft- 
rejection reactions result from the direct response of T cells 
to MHC molecules, which function as histocompatibility 
antigens. Because of the extreme polymorphism of the 
MHC, most individuals of the same species have unique sets 
of MHC molecules, or histocompatibility antigens, and are 
considered to be allogeneic, a term used to describe geneti- 
cally different individuals of the same species (see Chapter 
21). Therefore, T cells respond even to allografts (grafts from 
members of the same species), and MHC molecules are con- 
sidered alio antigens. Generally, CD4 + T cells are alloreactive 
to class II alloantigens, and CD8 + T cells respond to class I 
alloantigens. 

The alloreactivity of T cells is puzzling for two reasons. 
First, the ability of T cells to respond to allogeneic histocom- 
patibility antigens alone appears to contradict all the evi- 
dence indicating that T cells can respond only to foreign 
antigen plus seZ/-MHC molecules. In responding to allo- 
geneic grafts, however, T cells recognize a foreign MHC mol- 
ecule directly. A second problem posed by the T-cell response 
to allogeneic MHC molecules is that the frequency of allore- 
active T cells is quite high; it has been estimated that l%-5% 
of all T cells are reactive to a given alloantigen, which is 
higher than the normal frequency of T cells reactive with any 
particular foreign antigenic peptide plus self-MHC mole- 
cule. This high frequency of alloreactive T cells appears to 
contradict the basic tenet of clonal selection. If 1 T cell in 20 
reacts with a given alloantigen and if one assumes there are 
on the order of 100 distinct H-2 haplotypes in mice, then 
there are not enough distinct T-cell specificities to cover all 
the unique H-2 alloantigens, let alone foreign antigens dis- 
played by self-MHC molecules. 

One possible and biologically satisfying explanation for 
the high frequency of alloreactive T cells is that a particular 
T-cell receptor specific for a foreign antigenic peptide plus 
a self-MHC molecule can also cross-react with certain allo- 
geneic MHC molecules. In other words, if an allogeneic 
MHC molecule plus allogeneic peptide structurally resem- 
bles a processed foreign peptide plus self-MHC molecule, 
the same T-cell receptor may recognize both peptide-MHC 
complexes. Since allogeneic cells express on the order of 10 5 
class I MHC molecules per cell, T cells bearing low-affinity 
cross-reactive receptors might be able to bind by virtue of 
the high density of membrane alloantigen. Foreign antigen, 
on the other hand, would be sparsely displayed on the 
membrane of an antigen-presenting cell or altered self-cell 
associated with class I or class II MHC molecules, limiting 
responsiveness to only those T cells bearing high-affinity 
receptors. 



Information relevant to mechanisms for alloreactivity was 
gained by Reiser and colleagues, who determined the struc- 
ture of a mouse TCR complexed with an allogeneic class I 
molecule containing a bound octapeptide. This analysis re- 
vealed a structure similar to those reported for TCR bound to 
class I self-MHC complexes, leading the authors to conclude 
that allogeneic recognition is not unlike recognition of self- 
MHC antigens. The absence of negative selection for the pep- 
tides contained in the foreign MHC molecules can 
contribute to the high frequency of alloreactive T cells. This 
condition, coupled with the differences in the structure of 
the exposed portions of the allogeneic MHC molecule, may 
account for the phenomenon of alloreactivity. An explana- 
tion for the large number of alloreactive cells can be found in 
the large number of potential antigens provided by the for- 
eign molecule plus the possible peptide antigens bound by 



SUMMARY 

■ Most T-cell receptors, unlike antibodies, do not react with 
soluble antigen but rather with processed antigen bound 
to a self-MHC molecule; certain yS receptors recognize 
antigens not processed and presented with MHC. 

■ T-cell receptors, first isolated by means of clonotypic mon- 
oclonal antibodies, are heterodimers consisting of an a 
and (3 chain or a y and 8 chain. 

■ The membrane-bound T-cell receptor chains are orga- 
nized into variable and constant domains. TCR domains 
are similar to those of immunoglobulins and the V region 
has hypervariable regions. 

■ TCR germ-line DNA is organized into multigene families 
corresponding to the a, p, y, and 8 chains. Each family 
contains multiple gene segments. 

■ The mechanisms that generate TCR diversity are generally 
similar to those that generate antibody diversity, although 
somatic mutation does not occur in TCR genes, as it does 
in immunoglobulin genes. 

■ The T-cell receptor is closely associated with the CD3, 
a complex of polypeptide chains involved in signal 
transduction. 

■ T cells express membrane molecules, including CD4, CD8, 
CD2, LFA-1, CD28, and CD45R, that play accessory roles 
in T-cell function or signal transduction. 

■ Formation of the ternary complex TCR-antigen-MHC re- 
quires binding of a peptide to the MHC molecule and 
binding of the complex by the T-cell receptor. 

■ Interactions between TCR and MHC class I/peptide differ 
from those with MHC class II/peptide in the contact 
points between the TCR and MHC molecules. 

■ The y8 T-cell receptor is distinguished by ability to bind 
native antigens and by differences in the orientation of the 
variable and constant regions. 



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8536d:Goldsby et al . / ] 



■ In addition to reaction with self MHC plus foreign ai 
gens,T cells also respond to foreign MHC molecules, a 
action that leads to rejection of allogeneic grafts. 

References 



Study Questions 



1178 T-cell antiger 



Allison, T. J., et al. 2001. Structure c 
receptor. Nature 41 1:820. 

Gao, G. E, et al. 1997. Crystal structure of the complex between 
human CD8aa and HLA-A2. Nature 387:630. 

Garboczi, D. N„ et al. 1996. Structure of the complex between 
human T-cell receptor, viral peptide, and HLA-A2. Nature 
384:134. 

Garcia, K. C, et al. 1996. An a(3 T-cell receptor structure at 2.5 A 
and its orientation in the TCR-MHC complex. Science 
274:209. 

Garcia, K. C, et al. 1998. T-cell receptor-peptide-MHC interac- 
tions: biological lessons from structural studies. Curr. Opinions 
in Biotech. 9:338. 



and a right place for a 
Ann. Rev. Immunol. 



Hayday, A. 2000. 78 Cells: A right tin 
conserved third way of protectio 
18:1975. 

Hennecke J., and D. C. Wiley 2001. T-cell receptor-MHC inter- 
actions up close. Cell 104:1. 

Kabelitz, D., et al. 2000. Antigen recognition by 78 T lympho- 
cytes. Int. Arch. Allergy Immunol. 122: 1 . 

Reinherz, E., et al. 1999. The crystal structure of a T-cell receptor 
in complex with peptide and MHC class II. Science 286: 19 13. 

Reiser, J-B., et al. 2000. Crystal structure of a T-cell receptor 
bound to an allogeneic MHC molecule. Nature Immunology 
1:291. 

Sklar, J., et al. 1988. Applications of antigen-receptor gene re- 
arrangements to the diagnosis and characterization of lym- 
phoid neoplasms. Ann. Rev. Med. 39:315. 

Xiong, Y., et al. 2001. T-cell receptor binding to a pMHCII ligand 
is kinetically distinct from and independent of CD4. /. Biol. 
Chem. 276:5659. 

Zinkernagel, R. M., and P. C. Doherty. 1974. Immunological sur- 
veillance against altered self-components by sensitized T lym- 
phocytes in lymphocytic choriomeningitis. Nature 251:547. 



http://imgt.cines.fr 

A comprehensive database of genetic information on TCRs, 
MHC molecules, and immunoglobulins, from the Interna- 
tional ImmunoGenetics Database, University of Montpelier, 

http://www.bioscience.org/knockout/tcrab.htm 

This location presents a brief summary of the effects of TCR 
knockouts. 



Clinical Focus Question A patient presents with an enlarged 
lymph node, and a T-cell lymphoma is suspected. However, DNA 
sampled from biopsied tissue shows no evidence of a predomi- 
nant gene rearrangement when probed with a and (3 TCR genes. 
What should be done next to rule out lymphocyte malignancy? 

1 . Indicate whether each of the following statements is true or 
false. If you think a statement is false, explain why. 

a. Monoclonal antibody specific for CD4 will coprecipitate 
the T-cell receptor along with CD4. 

b. Subtractive hybridization can be used to enrich for mRNA 
that is present in one cell type but absent in another cell 
type within the same species. 

c. Clonotypic monoclonal antibody was used to isolate the 
T-cell receptor. 

d. The T cell uses the same set of V, D, and J gene segments as 
the B cell but uses different C gene segments. 

e. The ap TCR is bivalent and has two antigen-binding sites. 

f. Each ap T cell expresses only one P-chain and one a-chain 
allele. 

g. Mechanisms for generation of diversity of T-cell receptors 
are identical to those used by immunoglobulins. 

h. The Ig-a/Ig-P heterodimer and CD3 serve analogous 
functions in the B-cell receptor and T-cell receptor, 
respectively. 

2. What led Zinkernagel and Doherty to conclude that T-cell 
receptor recognition requires both antigen and MHC 
molecules? 



3. Draw the basic structure of the ap T-cell receptor and com- 
pare it with the basic structure of membrane-bound im- 
munoglobulin. 

4. Several membrane molecules, in addition to the T-cell recep- 
tor, are involved in antigen recognition and T-cell activation. 
Describe the properties and distinct functions of the follow- 
ing T-cell membrane molecules: (a) CD3, (b) CD4 and CD8, 
and (c) CD2. 

5. Indicate whether each of the properties listed below applies to 
the T-cell receptor (TCR), B-cell immunoglobulin (Ig), or 
both (TCR/Ig). 

a. Is associated with CD3 

b. Is monovalent 

c. Exists in membrane-bound and secreted forms 

d. Contains domains with the immunoglobulin-fold 



Is MHC restricted 

Exhibits diversity generated by imprecise joining 

of gene segments 

Exhibits diversity generated by somatic mutation 



6. A major obstacle to identifying and cloning TCR genes is the 
low level of TCR mRNA in T cells. 

a. To overcome this obstacle, Hedrick and Davis made three 
important assumptions that proved to be correct. Describe 
each assumption and how it facilitated identification of 
the genes that encode the T-cell receptor. 



Gotc 



/ww.whfreeman.cor 
and quiz of key ten 



no-logy 



KA Self-Test 



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ofB-CeilandT-Cell Respons 



b. Suppose, instead, that Hedrick and Davis wanted to iden- 
tify the genes that encode IL-4. What changes in the three 
assumptions should they make? 

7. Hedrick and Davis used the technique of subtractive hy- 
bridization to isolate cDNA clones that encode the T-cell re- 
ceptor. You wish to use this technique to isolate cDNA clones 
that encode several gene products and have available clones of 
various cell types to use as the source of cDNA or mRNA for 
hybridization. For each gene product listed in the left column 
of the table below, select the most appropriate. 



Gene 

product cDNA source mRNA source 


IL-2 






CD8 






J chain 






IL-1 






CD3 







cDNA and mRNA source clones are from the following cell 
types: T H 1 cell line (A); T H 2 cell line (B); T c cell line (C); 
macrophage (D); IgA-secreting myeloma cell (E); IgG-secret- 
ing myeloma cell (F); myeloid progenitor cell (G); and B-cell 
line (H). More than one cell type may be correct in some 



. Mice from different inbred strains listed in the left column of 
the accompanying table were infected with LCM virus. Spleen 
cells derived from these LCM-infected mice were then tested 
for their ability to lyse LCM-infected 51 Cr-labeled target cells 
from the strains listed across the top of the table. Indicate with 
( + ) or ( — ) whether you would expect to see 51 Cr released 
from the labeled target cells. 



cells from 
LCM-infected 


Release of 51 Crfrom 
LCM-infected target cells 


B10.D2 
(H-2 rf ) 


BIO 
(H-2 fc ) 


B10.BR 
(H-2 k ) 


(BALB/c X B10) F, 
[H-2 b/d ] 


B10.D2 
(H-2 rf ) 










BIO 

(H-2 fc ) 










BALB/c 










BALB/b 
(H-2 h ) 











9. The 78 T-cell receptor differs from the a(3 in both structural 
and functional parameters. Describe how they are similar to 
one another and different from the B-cell antigen receptors. 



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T-Cell Maturation, 
Activation, and 
Differentiation 



THE ATTRIBUTE THAT DISTINGUISHES ANTIGEN 
recognition by most T cells from recognition by B 
cells is MHC restriction. In most cases, both the 
maturation of progenitor T cells in the thymus and the acti- 
vation of mature T cells in the periphery are influenced by 
the involvement of MHC molecules. The potential antigenic 
diversity of the T-cell population is reduced during matura- 
tion by a selection process that allows only MHC-restricted 
and nonself-reactive T cells to mature. The final stages in the 
maturation of most T cells proceed along two different de- 
velopmental pathways, which generate functionally distinct 
CD4 + and CD8 + subpopulations that exhibit class II and 
class I MHC restriction, respectively. 

Activation of mature peripheral T cells begins with the 
interaction of the T-cell receptor (TCR) with an antigenic 
peptide displayed in the groove of an MHC molecule. Al- 
though the specificity of this interaction is governed by the 
TCR, its low avidity necessitates the involvement of corecep- 
tors and other accessory membrane molecules that 
strengthen the TCR-antigen-MHC interaction and trans- 
duce the activating signal. Activation leads to the prolifera- 
tion and differentiation of T cells into various types of 
effector cells and memory T cells. Because the vast majority 
of thymocytes and peripheral T cells express the a(3 T-cell 
receptor rather than the 78 T-cell receptor, all references to 
the T-cell receptor in this chapter denote the a (3 receptor un- 
less otherwise indicated. Similarly, unless otherwise indi- 
cated, all references to T cells denote those ap receptor- 
bearing T cells that undergo MHC restriction. 



T-Cell Maturation and the Thymus 

Progenitor T cells from the early sites of hematopoiesis begin 
to migrate to the thymus at about day 1 1 of gestation in mice 
and in the eighth or ninth week of gestation in humans. In a 
manner similar to B-cell maturation in the bone marrow, T- 
cell maturation involves rearrangements of the germ-line 
TCR genes and the expression of various membrane mark- 
ers. In the thymus, developing T cells, known as thymocytes, 
proliferate and differentiate along developmental pathways 
that generate functionally distinct subpopulations of mature 
T cells. 



chapter 10 




■ T-Cell Maturation and the Thymus 

■ Thymic Selection of the T-Cell Repertoire 

■ T H -Cell Activation 

■ T-Cell Differentiation 

■ Cell Death and T-Cell Populations 

■ Peripheral 78 T-Cells 



As indicated in Chapter 2, the thymus occupies a central 
role in T-cell biology. Aside from being the main source of all 
T cells, it is where T cells diversify and then are shaped into an 
effective primary T-cell repertoire by an extraordinary pair of 
selection processes. One of these, positive selection, permits 
the survival of only those T cells whose TCRs are capable of 
recognizing self-MHC molecules. It is thus responsible for 
the creation of a self-MHC-restricted repertoire of T cells. 
The other, negative selection, eliminates T cells that react 
too strongly with self-MHC or with self-MHC plus self- 
peptides. It is an extremely important factor in generating 
a primary T-cell repertoire that is self-tolerant. 

As shown in Figure 10-1, when T-cell precursors arrive at 
the thymus, they do not express such signature surface mark- 
ers of T cells as the T-cell receptor, the CD3 complex, or the 
coreceptors CD4 and CD8. In fact, these progenitor cells have 



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ofB-CeilandT-Cell Respons 



B^y^) VISUALIZING CO 

l 3P Development of a(3 
T cells in the mouse. T-cell precursors 
arrive at the thymus from bone mar- 

velopment to mature T cells, and are 
exported to the periphery where they 
can undergo antigen-induced activa- 
tion and differentiation into effector 
cells and memory cells. Each stage 
of development is characterized by 
stage-specific intracellular events and 
the display of distinctive cell-surface 


UEPTS 






Surface markers 




Marrow 
Blood 

Thymus 

Blood 
Peripheral 




— Hematopoietic 

f(~J\ stem cell C -Kit CD25 
^/ (HSC) CD44 

1 

— Common 

(C J) ly m phoid 

— precursor 






migration 










T_ceU 

vV-^y precursor 
TCR locus 
rearrangement ^^^ Pro . T cell 

r DfrJ|i (O) ( doubie 

V — / negative, DN) 

RAG ^.^ Pre-T cell 
expression- Vp-Dp-Jp (QV (double CI 
on ^ — ' negative, DN) 

Vp-Dp-Jp ^-Pro-Tcell 
1— and QK (double 
y t v — ' positive, DP) 

"V \ 

or + ©£ D " 


)3 




tcr|3 

Pre-T a 

TCR a ™4 
chain *° g 

1 
CD4 

CD8 


T c cell 

migration 












i i 
^— s. CD8+ , — s. CD4+ 













not yet rearranged their TCR genes and do not express pro- 
teins, such as RAG-1 and RAG-2, that are required for re- 
arrangement. After arriving at the thymus, these T-cell 
precursors enter the outer cortex and slowly proliferate. Dur- 
ing approximately three weeks of development in the thy- 
mus, the differentiating T cells progress through a series of 
stages that are marked by characteristic changes in their cell- 
surface phenotype. For example, as mentioned previously, 
thymocytes early in development lack detectable CD4 and 
CD8. Because these cells are CD4~CD8 - , they are referred to 
as double-negative (DN) cells. 

Even though these coreceptors are not expressed during 
the DN early stages, the differentiation program is progress- 
ing and is marked by changes in the expression of such cell 
surface molecules as c-Kit, CD44, and CD25. The initial thy- 
mocyte population displays c-Kit, the receptor for stem-cell 
growth factor, and CD44, an adhesion molecule involved in 
homing; CD25, the (3-chain of the IL-2 receptor, also appears 



on early-stage DN cells. During this period, the cells are pro- 
liferating but the TCR genes remain unrearranged. Then the 
cells stop expressing c-Kit, markedly reduce CD44 expres- 
sion, turn on expression of the recombinase genes RAG-1 
and RAG-2 and begin to rearrange their TCR genes. Al- 
though it is not shown in Figure 10-1, a small percentage 
(<5%) of thymocytes productively rearrange the y- and 
8-chain genes and develop into double-negative CD3 + y5 
T cells. In mice, this thymocyte subpopulation can be detected 
by day 14 of gestation, reaches maximal numbers between 
days 17 and 18, and then declines until birth (Figure 10-2). 

Most double-negative thymocytes progress down the a[3 
developmental pathway. They stop proliferating and begin to 
rearrange the TCR (3-chain genes, then express the (3 chain. 
Those cells of the a(3 lineage that fail to productively re- 
arrange and express (3 chains die. Newly synthesized (3 chains 
combine with a 33-kDa glycoprotein known as the pre-Ta 
chain and associate with the CD3 group to form a novel com- 



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3 mac76:385 ,1 




e course of appearance of 78 thyrr 
af3 thymocytes during mouse fetal development. The graph shows 
the percentage of CD3 + cells in the thymus that are double-negative 
(CD4~8~) and bear the 78 T-cell receptor (black) or are double- 
positive (CD4 + 8 + ) and bear the a(3 T-cell receptor (blue). 



plex called the pre-T-cell receptor or pre-TCR (Figure 10-3). 
Some researchers have suggested that the pre-TCR recog- 
nizes some intra-thymic ligand and transmits a signal 
through the CD3 complex that activates signal-transduction 
pathways that have several effects: 

■ Indicates that a cell has made a productive TCR p-chain 
rearrangement and signals its further proliferation and 



Cell becomes 
permissive for 
TCR a-chain locus 
arrangement 




Stops additional 
TCR (3-chain locus 
arrangements 
(allelic exclusion) 



expression 
of CD4 and 
CD8 coreceptors 



•J Structure and activity of the pre-T-cell receptor (pre- 
TCR). Binding of ligands yet to be identified to the pre-TCR ger 
intracellular signals that induce a variety of processes. 



■ Suppresses further rearrangement of TCR p-chain genes, 
resulting in allelic exclusion. 

■ Renders the cell permissive for rearrangement of the 
TCR a chain. 

■ Induces developmental progression to the CD4 + 8 + 
double-positive state. 

After advancing to the double-positive (DP) stage, where 
both CD4 and CD8 coreceptors are expressed, the thymo- 
cytes begin to proliferate. However, during this proliferative 
phase, TCR a-chain gene rearrangement does not occur; 
both the RAG-1 and RAG-2 genes are transcriptionally ac- 
tive, but the RAG-2 protein is rapidly degraded in proliferat- 
ing cells, so rearrangement of the a-chain genes cannot take 
place. The rearrangement of a-chain genes does not begin 
until the double-positive thymocytes stop proliferating and 
RAG-2 protein levels increase. The proliferative phase prior 
to the rearrangement of the a-chain increases the diversity of 
the T-cell repertoire by generating a clone of cells with a sin- 
gle TCR (3-chain rearrangement. Each of the cells within this 
clone can then rearrange a different a-chain gene, thereby 
generating a much more diverse population than if the orig- 
inal cell had first undergone rearrangement at both the (3- 
and a-chain loci before it proliferated. In mice, the TCR a- 
chain genes are not expressed until day 16 or 17 of gestation; 
double-positive cells expressing both CD3 and the a(3 T-cell 
receptor begin to appear at day 17 and reach maximal levels 
about the time of birth (see Figure 10-2). The possession of a 
complete TCR enables DP thymocytes to undergo the rigors 
of positive and negative selection. 

T-cell development is an expensive process for the host. 
An estimated 98% of all thymocytes do not mature — they 
die by apoptosis within the thymus either because they fail to 
make a productive TCR-gene rearrangement or because they 
fail to survive thymic selection. Double-positive thymocytes 
that express the a(3 TCR-CD3 complex and survive thymic 
selection develop into immature single-positive CD4 + 
thymocytes or single-positive CD8 + thymocytes. These 
single-positive cells undergo additional negative selection 
and migrate from the cortex to the medula, where they pass 
from the thymus into the circulatory system. 



Thymic Selection of the 
T-Cell Repertoire 

Random gene rearrangement within TCR germ-line DNA 
combined with junctional diversity can generate an enor- 
mous TCR repertoire, with an estimated potential diversity 
exceeding 10 15 for the a(3 receptor and 10 18 for the ~y8 recep- 
tor. Gene products encoded by the rearranged TCR genes have 
no inherent affinity for foreign antigen plus a self-MHC mol- 
ecule; they theoretically should be capable of recognizing sol- 
uble antigen (either foreign or self), self-MHC molecules, or 



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8/29/02 10:23 AM Page 224 



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ofB-CeilandT-Cell Respons 



n plus a nonself-MHC molecule. Nonetheless, the most 
e property of mature T cells is that they recognize 
only foreign antigen combined with self-MHC molecules. 

As noted, thymocytes undergo two selection processes in 
the thymus: 

■ Positive selection for thymocytes bearing receptors 
capable of binding self-MHC molecules, which results in 
MHC restriction. Cells that fail positive selection are 
eliminated within the thymus by apoptosis. 

■ Negative selection that eliminates thymocytes bearing 
high-affinity receptors for self-MHC molecules alone or 
self-antigen presented by self-MHC, which results in 
self-tolerance. 



Both processes are necessary to generate mature T cells that 
are self-MHC restricted and self-tolerant. As noted already, 
some 98% or more of all thymocytes die by apoptosis within 
the thymus. The bulk of this high death rate appears to reflect 
a weeding out of thymocytes that fail positive selection be- 
cause their receptors do not specifically recognize foreign 
antigen plus self-MHC molecules. 

Early evidence for the role of the thymus in selection of 
the T-cell repertoire came from chimeric mouse experi- 
ments by R. M. Zinkernagel and his colleagues (Figure 
10-4). These researchers implanted thymectomized and ir- 
radiated (A X B) ¥i mice with a B-type thymus and then 
reconstituted the animal's 
venous infusion of Fj bone- 
the thymus graft did 
irradiated before being transplai 
mental system, T-cell pro 
bone-marrow transplant n 
presses only B-haplotype MHC molecules on its stromal 
cells. Would these (A X B) Fj T cells now be MHC- 
restricted for the haplotype of the thymus? To answer this 
question, the chimeric mice were infected with LCM virus 
and the immature T cells were then tested for their ability to 
kill LCM-infected target cells from the strain A or strain B 
mice. As shown in Figure 10-4, when T c cells from the 
chimeric mice were tested on LCM virus infected target 
cells from strain A or strain B mice, they could only lyse 
LCM-infected target cells from strain B mice. These mice 
have the same MHC haplotype, B, as the implanted thymus. 
Thus, the MHC haplotype of the thymus in which T cells 
develop determines their MHC restriction. 

Thymic stromal cells, including epithelial cells, macro- 
phages, and dendritic cells, play essential roles in positive and 
negative selection. These cells express class I MHC molecules 
and can display high levels of class II MHC also. The interac- 
tion of immature thymocytes that express the TCR-CD3 
complex with populations of thymic stromal cells results in 
positive and negative selection by mechanisms that are under 
intense investigation. First, we'll examine the details of each 
selection process and then study some experiments that pro- 
vide insights into the operation of these pre 



EXPERIMENT 



system with an intra- 
cells. To be certain that 
my mature T cells, it was 
ted. In such an experi- 
s from the (A X B) Fj 
within a thymus that ex- 



x B)F! (H-2«/6) 

(T) Thymectomy 

@ Lethal x-irradiation 




Strain-B thymus graft (H-2 6 ) 
(A x B)Fj hematopoietic stem 
cells (H-2"/ fi ) 

ith LCM virus 



n LCM-infected n 
{{ strain-A cells J 


1 


LCM-infected n 


1 


strain-B cells J 


No killing 




Killing 


CONTROL 







- Infect with LCM v 









| Experimental demonstration that the thymus selects 
for maturation only those T cells whose T-cell receptors recognize 
antigen presented on target cells with the haplotype of the thymus. 
Thymectomized and lethally irradiated (A X B) F, mice were grafted 
with a strain-B thymus and reconstituted with (A X B) F, bone- 
marrow cells. After infection with the LCM virus, the CTL cells were 
assayed for their ability to kill 51 Cr-labe!ed strain-A or strain-B target 
cells infected with the LCM virus. Only strain-B target cells were 
lysed, suggesting that the \r\-2 b grafted thymus had selected for 
maturation only those T cells that could recognize antigen combined 
with H-2 b MHC molecules. 



Positive Selection Ensures MHC Restriction 

Positive selection takes place in the cortical region of the thy- 
mus and involves the interaction of immature thymocytes 
with cortical epithelial cells (Figure 10-5). There is evidence 
that the T-cell receptors on thymocytes tend to cluster with 



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:76 mac76:385 ,1 



Rearrangement of TCR genes 




Negative selection and death of 
"inity receptors 
for self-MHC or self-MHC + 



T„ cell T, 
Mature CD4+ 
CD8+ T lymphocyK 




thymus. Thymic selection involves thyr 
cells, dendritic cells, and macrophages 
that are both self-MHC restricted and 



MHC molecules on the cortical cells at sites of cell-cell con- 
tact. Some researchers have suggested that these interactions 
allow the immature thymocytes to receive a protective signal 
that prevents them from undergoing cell death; cells whose 
receptors are not able to bind MHC molecules would not in- 
teract with the thymic epithelial cells and consequently 
would not receive the protective signal, leading to their death 
by apoptosis. 



During positive selection, the RAG-1, RAG-2, and TdT 
proteins required for gene rearrangement and modification 
continue to be expressed. Thus each of the immature thymo- 
cytes in a clone expressing a given (3 chain have an opportu- 
nity to rearrange different TCR a-chain genes, and the 
resulting TCRs are then selected for self-MHC recognition. 
Only those cells whose ap TCR heterodimer recognizes a 
self-MHC molecule are selected for survival. Consequently, 
the presence of more than one combination of a (3 TCR 
chains among members of the clone is important because it 
increases the possibility that some members will "pass" the 
test for positive selection. Any cell that manages to rearrange 
an a chain that allows the resulting ap TCR to recognize self- 
MHC will be spared; all members of the clone that fail to do 
so will die by apoptosis within 3 to 4 days. 

Negative Selection Ensures Self-Tolerance 

The population of MHC-restricted thymocytes that survive 
positive selection comprises some cells with low-affinity re- 
ceptors for self-antigen presented by self-MHC molecules 
and other cells with high-affinity receptors. The latter thy- 
mocytes undergo negative selection by an interaction with 
thymic stromal cells. During negative selection, dendritic 
cells and macrophages bearing class I and class II MHC mol- 
ecules interact with thymocytes bearing high-affinity recep- 
tors for self-antigen plus self-MHC molecules or for 
self-MHC molecules alone (see Figure 10-5). However, the 
precise details of the process are not yet known. Cells that ex- 
perience negative selection are observed to undergo death by 
apoptosis. Tolerance to self-antigens encountered in the thy- 
mus is thereby achieved by eliminating T cells that are reac- 
tive to these antigens. 

Experiments Revealed the Essential Elements 
of Positive and Negative Selection 

Direct evidence that binding of thymocytes to class I or class 
II MHC molecules is required for positive selection in the 
thymus came from experimental studies with knockout mice 
incapable of producing functional class I or class II MHC 
molecules (Table 10-1). Class I-deficient mice were found to 
have a normal distribution of double-negative, double-posi- 
tive, and CD4 + thymocytes, but failed to produce CD8 + thy- 
mocytes. Class II-deficient mice had double-negative, 
double-positive, and CD8 + thymocytes but lacked CD4 + 
thymocytes. Not surprisingly, the lymph nodes of these class 
II-deficient mice lacked CD4 + T cells. Thus, the absence of 
class I or II MHC molecules prevents positive selection of 
CD8 + or CD4 + T cells, respectively. 

Further experiments with transgenic mice provided addi- 
tional evidence that interaction with MHC molecules plays a 
role in positive selection. In these experiments, rearranged 
ct(3-TCR genes derived from a CD8 + T-cell clone specific for 
influenza antigen plus H-2* class I MHC molecules were in- 
jected into fertilized eggs from two different mouse strains, 



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ofB-CellandT-Cell Respons 



TABLE 10-1 



KNOCKOUT MICE 

Control Class I Class II 

Cell type mice deficient deficient 

CD4~CD8~ + + + 

CD4 + CD8 + + + + 

CD4 + + + 
CD8 + + 



one with the H-2 fc haplotype and one with the H-2 d haplo- 
type (Figure 10-6). Since the receptor transgenes were al- 
ready rearranged, other TCR-gene rearrangements were 
suppressed in the transgenic mice; therefore, a high percent- 
age of the thymocytes in the transgenic mice expressed the 
T-cell receptor encoded by the transgene. Thymocytes 
expressing the TCR transgene were found to mature into 
CD8 + T cells only in the transgenic mice with the H-2* class 
I MHC haplotype (i.e., the haplotype for which the transgene 
receptor was restricted). In transgenic mice with a different 
MHC haplotype (H-2 d ), immature, double-positive thymo- 
cytes expressing the transgene were present, but these thy- 
mocytes failed to mature into CD8 + T cells. These findings 
confirmed that interaction between T-cell receptors on im- 
mature thymocytes and self-MHC molecules is required for 
positive selection. In the absence of self-MHC molecules, as 
in the H-2 d transgenic mice, positive selection and subse- 
quent maturation do not occur. 

Evidence for deletion of thymocytes reactive with self- 
antigen plus MHC molecules comes from a number of ex- 
perimental systems. In one system, thymocyte maturation 
was analyzed in transgenic mice bearing an ap TCR trans- 
gene specific for the class I D b MHC molecule plus H-Y anti- 
gen, a small protein that is encoded on the Y chromosome 
and is therefore a self-molecule only in male mice. In this ex- 
periment, the MHC haplotype of the transgenic mice was 
H-2 b , the same as the MHC restriction of the transgene- 
encoded receptor. Therefore any differences in the selection 
of thymocytes in male and female transgenics would be re- 
lated to the presence or absence of H-Y antigen. 

Analysis of thymocytes in the transgenic mice revealed 
that female mice contained thymocytes expressing the H-Y- 
specific TCR transgene, but male mice did not (Figure 10-7). 
In other words, H-Y-reactive thymocytes were self-reactive 
in the male mice and were eliminated. However, in the female 
transgenics, which did not express the H-Y antigen, these 
cells were not self-reactive and thus were not eliminated. 
When thymocytes from these male transgenic mice were cul- 



tured in vitro with antigen-presenting cells expressing the 
H-Y antigen, the thymocytes were observed to undergo 
apoptosis, providing a striking example of negative selection. 

Some Central Issues in Thymic Selection 
Remain Unresolved 

Although a great deal has been learned about the develop- 
mental processes that generate mature CD4 and CD8 T 
cells, some mysteries persist. Prominent among them is a 
seeming paradox: If positive selection allows only thymo- 
cytes reactive with self-MHC molecules to survive, and nega- 
tive selection eliminates the self-MHC-reactive thymo- 
cytes, then no T cells would be allowed to mature. Since this 
is not the outcome of T-cell development, clearly, other fac- 
tors operate to prevent these two MHC-dependent processes 
from eliminating the entire repertoire of MHC-restricted T 
cells. 

Experimental evidence from fetal thymic organ culture 
(FTOC) has been helpful in resolving this puzzle. In this sys- 
tem, mouse thymic lobes are excised at a gestational age of day 
16 and placed in culture. At this time, the lobes consist pre- 
dominantly of CD4~8~ thymocytes. Because these immature, 
double-negative thymocytes continue to develop in the organ 
culture, thymic selection can be studied under conditions that 
permit a range of informative experiments. Particular use has 






H-2* 


H-2'' 






transgenic 


in transgenics 






TCR + /CD4 + 8 + 


+ 


+ 


TCR+/CD8+ 


+ 


- 



Effect of host haplotype on T-cell maturation in mice 
carrying transgenes encoding an H-2 b class l-restricted T-cell recep- 
tor specific for influenza virus. The presence of the rearranged TCR 
transgenes suppressed other gene rearrangements in the transgen- 
ics; therefore, most of the thymocytes in the transgenics expressed 
the a(3 T-cell receptor encoded by the transgene. Immature double- 
positive thymocytes matured into CD8 + T cells only in transgenics 
with the haplotype (H-2 ,< ) corresponding to the MHC n 
the TCR transgene. 



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3 mac76:385 ,1 






Male H-2D& 


Female H-2D b 


H-Y expression 


+ 


- 


Thymocytes 


CD4-8- 


+ + 


+ 


CD4+8+ 


+ 


+ + 


CD4+ 


+ 


+ 


CD8+ 


- 


+ + 



Experimental demonstration that 
negative selection of thymocytes requires self-anti- 
gen plus self-MHC. In this experiment, H-2 fc male 
and female transgenics were prepared carrying TCR 
transgenes specific for H-Y antigen plus the D b mol- 
ecule. This antigen is expressed only in males. FACS 
analysis of thymocytes from the transgenics showed 
that mature CD8 + T cells expressing the transgene 
were absent in the male mice but present in the fe- 
male mice, suggesting that thymocytes reactive with 
a self-antigen (in this case, H-Y antigen in the male 
mice) are deleted during thymic selection. [Adapted 
from H. von Boehmer and P. Kisielow, 1990, Science 
248:1370.] 



been made of mice in which the peptide transporter, TAP-1, 
has been knocked out. In the absence of TAP- 1 , only low levels 
of MHC class I are expressed on thymic cells, and the develop- 
ment of CD8 + thymocytes is blocked. However, when exoge- 
nous peptides are added to these organ cultures, then 
peptide-bearing class I MHC molecules appear on the surface 
of the thymic cells, and development of CD8 + T cells is re- 
stored. Significantly, when a diverse peptide mixture is added, 
the extent of CD8 + T-cell restoration is greater than when a 
single peptide is added. This indicates that the role of peptide 
is not simply to support stable MHC expression but also to be 
recognized itself in the selection process. 

Two competing hypotheses attempt to explain the para- 
dox of MHC-dependent positive and negative selection. The 
avidity hypothesis asserts that differences in the strength of 
the signals received by thymocytes undergoing positive and 
negative selection determine the outcome, with signal 
strength dictated by the avidity of the TCR-MHC-peptide in- 
teraction. The differential-signaling hypothesis holds that the 
outcomes of selection are dictated by different signals, rather 
than different strengths of the same signal. 

The avidity hypothesis was tested with TAP-1 knockout 
mice transgenic for an a (3 TCR that recognized an LCM virus 
peptide-MHC complex. These mice were used to prepare fe- 
tal thymic organ cultures (Figure 10-8). The avidity of the 
TCR-MHC interaction was varied by the use of different 



concentrations of peptide. At low peptide concentrations, 
few MHC molecules bound peptide and the avidity of the 
TCR-MHC interaction was low. As peptide concentrations 
were raised, the number of peptide-MHC complexes dis- 
played increased and so did the avidity of the interaction. In 
this experiment, very few CD8 + cells appeared when peptide 
was not added, but even low concentrations of the relevant 
peptide resulted in the appearance of significant numbers of 
CD8 + T cells bearing the transgenic TCR receptor. Increas- 
ing the peptide concentrations to an optimum range yielded 
the highest number of CD8 + T cells. However, at higher con- 
centrations of peptide, the numbers of CD8 + T cells pro- 
duced declined steeply. The results of these experiments 
show that positive and negative selection can be achieved 
with signals generated by the same peptide-MHC combina- 
tion. No signal (no peptide) fails to support positive selec- 
tion. A weak signal (low peptide level) induces positive 
selection. However, too strong a signal (high peptide level) 
results in negative selection. 

The differential-signaling model provides an alternative 
explanation for determining whether a T cell undergoes posi- 
tive or negative selection. This model is a qualitative rather 
than a quantitative one, and it emphasizes the nature of the 
signal delivered by the TCR rather than its strength. At the 
core of this model is the observation that some MHC-peptide 
complexes can deliver only a weak or partly activating signal 



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ofB-CeilandT-Cell Respons 



while others can deliver a complete signal. In this model, pos- 
itive selection takes place when the TCRs of developing thy- 
mocytes encounter MHC-peptide complexes that deliver 
weak or partial signals to their receptors, and negative selec- 
tion results when the signal is complete. At this point it is not 
possible to decide between the avidity model and the differen- 
tial-signaling model; both have experimental support. It may 
be that in some cases, one of these mechanisms operates to the 
complete exclusion of the other. It is also possible that no sin- 
gle mechanism accounts for all the outcomes in the cellular 
interactions that take place in the thymus and more than one 
mechanism may play a significant role. Further work is re- 
quired to complete our understanding of this matter. 

The differential expression of the coreceptor CD8 also can 
affect thymic selection. In an experiment in which CD8 ex- 






s artificially raised to twice its normal level, the 
nature CD8 + cells in the thymus was one- 
thirteenth of the concentration in control mice bearing nor- 
mal levels of CD8 on their surface. Since the interaction of T 
cells with class I MHC molecules is strengthened by partici- 
pation of CD8, perhaps the increased expression of CD8 
would increase the avidity of thymocytes for class I mole- 
cules, possibly making their negative selection more likely. 

Another important open question in thymic selection is 
how double-positive thymocytes are directed to become ei- 
ther CD4 + 8~ or CD4~8 + T cells. Selection of CD4 + 8 + thy- 
mocytes gives rise to class I MHC-restricted CD8 + T cells 
and class II-restricted CD4 + T cells. Two models have been 
proposed to explain the transformation of a double-positive 
precursor into one of two different single-positive lineages 



mental procedure — fetal thymic organ culture (FTOC) 



(b) Development of CD8 + CD4" MHC I-restricted cells 



x Growth medium 



of peptides in selection. 
Thymuses harvested before their thymocyte 
populations have undergone positive and 
negative selection allow study of the develop- 
ment and selection of single positive 
(CD4 + CD8~ and CD4~CD8 + ) T cells, (a) 
Outline of the experimental procedure for in 
vitro fetal thymic organ culture (FTOC). (b) 
The development and selection of 
CD8 + CD4~ class l-restricted T cells depends 
on TCR peptide-MHC I interactions. TAP,! 
knockout mice are unable to form peptide- 
MHC complexes unless peptide is added. 
The mice used in this study were transgenic 
for the a and |3 chains of a TCR that recog- 
nizes the added peptide bound to MHC I 
molecules of the TAP., knockout/TCR 
genie mice. Varying the amount of added pep- 
tide revealed that low concentrations 
peptide, producing low avidity of binding, 
suited in positive selection and nearly nor 
levels of CD4~CD8 + cells. High i 
tions of peptide, producing high 
binding to the TCR, caused negative selection, 
and few CD4~CD8 + T cells appeared. 
[Adapted from Ashton Rickardt et al. (1994) 
Cell 25:651.] 







Thymocyte ^^ 


Degree of 


Thymus 


Amount of 


^ - __-—-""~'^ Thymic 


CD8 + T-cell 


donor 


peptide added 


stromal cell 


development 



Normal 


Peptide -"" 


^ Weak signal 


Normal 


TCR-transgenic 
TAP-1 deficient 




x-> No signal 


Minimal 




~ : 


^ Weak signal 


Approaches 
normal 




High 


^-> Strong signal 


Minimal 



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4- CD4 
CD4+8+ CD4'»8 



CD4-8+Tcell 



CD4+8-TceU 



Proposed models for the role 
of the CD4 and CD8 coreceptors in thymic se- 
lection of double positive thymocytes leading 
to single positive T cells. According to the in- 
structive model, interaction of one coreceptor 
with MHC molecules on stromal cells results 
in down-regulation of the other coreceptor. 
According to the stochastic model, down- 
regulation of CD4 or CD8 is a random process. 



(Figure 10-9). The instructional model postulates that the 
multiple interactions between the TCR, CD8 + or CD4 + 
coreceptors, and class I or class II MHC molecules instruct 
the cells to differentiate into either CD8 + or CD4 + single- 
positive cells, respectively. This model would predict that a 
class I MHC-specific TCR together with the CD8 coreceptor 
would generate a signal that is different from the signal in- 
duced by a class II MHC-specific TCR together with the 
CD4 coreceptor. The stochastic model suggests that CD4 or 
CD8 expression is switched off randomly with no relation to 
the specificity of the TCR. Only those thymocytes whose 
TCR and remaining coreceptor recognize the same class of 
MHC molecule will mature. At present, it is not possible to 
choose one model over the other. 



T H -Cell Activation 

The central event in the generation of both humoral and cell- 
mediated immune responses is the activation and clonal ex- 
pansion of T H cells. Activation of T c cells, which is generally 
similar to T H -cell activation, is described in Chapter 14. T H - 
cell activation is initiated by interaction of the TCR-CD3 
complex with a processed antigenic peptide bound to a class 
II MHC molecule on the surface of an antigen-presenting 
cell. This interaction and the resulting activating signals also 
involve various accessory membrane molecules on the T H 
cell and the antigen-presenting cell. Interaction of a T H cell 
with antigen initiates a cascade of biochemical events that in- 
duces the resting T H cell to enter the cell cycle, proliferating 



and differentiating into memory cells or effector cells. Many 
of the gene products that appear upon interaction with anti- 
gen can be grouped into one of three categories depending 
on how early they can be detected after antigen recognition 
(Table 10-2): 

■ Immediate genes, expressed within half an hour of 
antigen recognition, encode a number of transcription 
factors, including c-Fos, c-Myc, c-Jun, NFAT, and NF-kB 

■ Early genes, expressed within 1-2 h of antigen 
recognition, encode IL-2, IL-2R (IL-2 receptor), IL-3, 
IL-6, IFN-'v, and numerous other proteins 



Late genes, expressed n 
recognition, encode va 



e than 2 days after antigt 
us adhesion molecules 



These profound changes are the result of signal-transduction 
pathways that are activated by the encounter between the 
TCR and MHC-peptide complexes. An overview of some of 
the basic strategies of cellular signaling will be useful back- 
ground for appreciating the specific signaling pathways used 
by T cells. 

Signal-Transduction Pathways Have Several 
Features in Common 

The detection and interpretation of signals from the environ- 
ment is an indispensable feature of all cells, including those of 
the immune system. Although there are an enormous number 
of different signal-transduction pathways, some common 
themes are typical of these crucial integrative processes: 



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ofB-CellandT-Cell Respons 



| TABLE 10-2 ^ 


Gene product 


Function 


Time mRNA 
expression begins 


Location 


Ratio of activated to 
nonactivated cells 


IMMEDIATE 



NFAT 



Protooncogene; 
nuclear-binding protein 

transcription factor 
Transcription factor 
Cellular oncogene 
Transcription factor 



I L-2 receptor (p55) 

TNF-p 

Cyclin 



c-Myb 
GM-CSF 



Cytokin 
Cytokin 



ceptor 



Cytokine 

Cytokine 

Cytokine receptor 

Cytokine 

Cell-cycle protein 

Cytokine 

Cytokine 

Cytokine 

Protooncogene 

Cytokine 



Cell memb 
Cellmemb 

Secreted 
Secreted 
Nucleus 



HLA-DR 

VLA-4 

VLA-1,VLA-2,VLA-3, VLA-5 



II MHCmoleculi 
ion molecule 
ion molecules 



SOURCE: A 



id from C. Cral 



3-5 days 
7-14 days 



on begins with the interaction between a 
signal and its receptor. Signals that cannot penetrate the 
cell membrane bind to receptors on the surface of the 
cell membrane. This group includes water-soluble 
signaling molecules and membrane-bound ligands 
(MHC-peptide complexes, for example). Hydrophobic 
signals, such as steroids, that can diffuse through the cell 
membrane are bound by intracellular receptors. 

Signals are often transduced through G proteins, 
membrane-linked macromolecules whose activities are 
controlled by binding of the guanosine nucleotides GTP 
and GDP, which act as molecular switches. Bound GTP 
turns on the signaling capacities of the G protein; 



hydrolysis of GTP or exchange for GDP turns off the 
signal by returning the G protein to an inactive form. 
There are two major categories of G proteins. Small G 
proteins consist of a single polypeptide chain of about 
21 kDa. An important small G protein, known as Ras, 
is a key participant in the activation of an important 
proliferation-inducing signal-transduction cascade 
triggered by binding of ligands to their receptor tyrosine 
kinases. Large G proteins are composed of a, (3, and 7 
subunits and are critically involved in many processes, 
including vision, olfaction, glucose metabolism, and 
phenomena of immunological interest such as leukocyte 
chemotaxis. 



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T-Cell Maturation, Activ; 



Signal reception often leads to the generation within the 
cell of a "second messenger," a molecule or ion that can 
diffuse to other sites in the cell and evoke changes. 
Examples are cyclic nucleotides (cAMP, cGMP), calcium 
ion (Ca 2+ ), and membrane phospholipid derivatives 
such as diacylglycerol (DAG) and inositol triphosphate 
(IPs). 

Protein kinases and protein phosphatases are activated or 
inhibited. Kinases catalyze the phosphorylation of target 
residues (tyrosine, serine, or threonine) of key elements 
in many signal-transduction pathways. Phosphatases 
catalyze dephosphorylation, reversing the effect of 
kinases. These enzymes play essential roles in many 
signal-transduction pathways of immunological interest. 

Many signal transduction pathways involve the signal- 
induced assembly of some components of the pathway. 
Molecules known as adaptor proteins bind specifically 
and simultaneously to two or more different molecules 
with signaling roles, bringing them together and 
promoting their combined activity. 

Signals are amplified by enzyme cascades. Each enzyme in 
the cascade catalyzes the activation of many copies of the 
next enzyme in the sequence, greatly amplifying the 
signal at each step and offering many opportunities to 
modulate the intensity of a signal along the way. 

The default set, on pathways is 

OFF. In the absence of an appropriately presented signal, 
n through the pathway does not take place. 



Multiple Signaling Pathways Are Initiated 
byTCR Engagement 

The events that link antigen recognition by the T-cell recep- 
tor to gene activation echo many of the themes just reviewed. 
The key element in the initiation of T-cell activation is the 
recognition by the TCR of MHC-peptide complexes on 
antigen-presenting cells. 

As described in Chapter 9, the TCR consists of a mostly 
extracellular ligand-binding unit, a predominantly intracel- 
lular signaling unit, the CD3 complex, and the homodimer of 
t, (zeta) chains. Experiments with knockout mice have shown 
that all of these components are essential for signal transduc- 
tion. Two phases can be recognized in the antigen-mediated 
induction of T-cell responses: 

■ Initiation. The engagement of MHC-peptide by the TCR 
leads to clustering with CD4 or CD8 coreceptors as these 
coreceptors bind to invariant regions of the MHC 
molecule (Figure 10-10). Lck, a protein tyrosine kinase 
associated with the cytoplasmic tails of the coreceptors, 
is brought close to the cytoplasmic tails of the TCR 
complex and phosphorylates the immunoreceptor 
tyrosine-based activation motifs (ITAMs, described in 
Chapter 9). The phosphorylated tyrosines in the ITAMs 



of the zeta chain provide docking sites to which a protein 
tyrosine kinase called ZAP-70 attaches (step 2 in Figure 
10-10) and becomes active. ZAP-70 then catalyzes the 
phosphorylation of a number of membrane-associated 
adaptor molecules (step 3), which act as anchor points 
for the recruitment of several intracellular signal 
transduction pathways. One set of pathways involves a 
form of the enzyme phospholipase C (PLC), which 
anchors to an adaptor molecule, is activated by 
phosphorylation and cleaves a membrane phospholipid 
to generate second messengers. Another set activates 
small G proteins. 

■ Generation of multiple intracellular signals. Many 

signaling pathways are activated as a consequence of the 
steps that occur in the initiation phase, as shown to the 
right in Figure 10-10, and described below. 

We shall consider several of the signaling pathways re- 
cruited by T-cell activation, but the overall process is quite 
complex and many of the details will not be presented here. 
The review articles suggested at the end of this chapter pro- 
vide extensive coverage of this very active research area. 
Phospholipase C~y (PLCy): PLC 7 is activated by phosphoryla- 
tion and gains access to its substrate by binding to a mem- 
brane-associated adaptor protein (Figure 10-1 la). PLC7 
hydrolyzes a phospholipid component of the membrane to 
generate inositol 1,4,5-triphosphate (IP 3 ) and diacylglycerol 
(DAG). IP 3 causes a rapid release of Ca 2+ from the endoplas- 
mic reticulum and opens Ca 2+ channels in the cell mem- 
brane (Figure 10-llb). DAG activates protein kinase C, a 
multifunctional kinase that phosphorylates many different 
targets (Figure 10-1 lc). 

Ca 2+ : Calcium ion is involved in an unusually broad range of 
processes, including vision, muscle contraction, and many 
others. It is an essential element in many T-cell responses, in- 
cluding a pathway that leads to the movement of a major 
transcription factor, NFAT, from the cytoplasm into the nu- 
cleus (Figure 10-llb). In the nucleus, NFAT supports the 
transcription of genes required for the expression of the T- 
cell growth-promoting cytokines IL-2, IL-4, and others. 
Protein kinase C (PKC): This enzyme, which affects many 
pathways, causes the release of an inhibitory molecule from 
the transcription factor NF-kB, allowing NF-kB to enter the 
nucleus, where it promotes the expression of genes required 
for T-cell activation (Figure 10- lie). NF-kB is essential for a 
variety of T-cell responses and provides survival signals that 
protect T cells from apoptotic death. 

The Ras/MAP kinase pathway: Ras is a pivotal component of 
a signal-transduction pathway that is found in many cell 
types and is evolutionarily conserved across a spectrum of 
eukaryotes from yeasts to humans. Ras is a small G protein 
whose activation by GTP initiates a cascade of protein ki- 
nases known as the mitogen-activated protein kinase (MAP 
kinase) pathway. As shown in Figure 10-12, phosphorylation 
of the end product of this cascade, MAP kinase (also called 



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VISUALIZING CONCEPTS 



| Engagement of MHC-peptide initiates processes 
that lead to assembly of signaling complex. 




T CD4/8-associated Lck phosphorylates ITAMs of 
coreceptors, creates docking site for ZAP-70 




GADS LAT 



J ZAP-70 phosphorylates adaptor molecules that 
recruit components of several signaling pathways 



> Changes in gene expression 

> Functional changes 

itiation 



I Overview of TCR-mediated signs 
gagement by peptide-MHC complexes initiates the 
signaling complex. An early step is the Lck-mediate 
lation of ITAMs on the zeta (Q chains of the TCR o 



.ling. TCR en- 

;d phosphory- 

Dmplex, creat- 



ing docking sites to which the protein kinase ZAP-70 attaches 
and becomes activated by phosphorylation. A series of ZAP-70- 
catalyzed protein phosphorylations enable the generation of a 
variety of signals. (Abbreviations: DAG = diacylglycerol; CADS = 



Crb2-like adaptor downstream of She; GEF = guanine nucleotide 
exchange factor; ITAM = immunoreceptor tyrosine-based activa- 
tion motif; Itk = inducible T cell kinase; IP3 = inositol 1,4,5 
triphosphate; LAT = linker of activated T cells; PIP, = phospho- 
inositol biphosphage; PLC7 = phospholipase C gamma; Lck = 
lymphocyte kinase; SLP-76 = SH2-containing leukocyte-specific 
protein of 76 kDa; ZAP-70 = zeta associated protein of 70 kDa.) 



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3 mac76:385 a 



A A A A A A A A A A A A A 




calmodulin-Ca 2+ 

NFAT -@ — ^- — > NFAT 
® 



TTTTTTTTTTTTTTTTTTTTTTTTTT 
A A A A A A A A A A A A A A A A A A A A A A A A A A 



TTTTTTTT 
A A A A A A A A 



WW 
A A A A 




30000000000C 

Transcriptional a< 
of several genes 






Signal-transduc 
ways associated with T-cell 
(a) PhospholipaseC-y (PLC) is activated by 
phosphorylation. Active PLC hydrolyzes a 
phospholipid component of the plasma 
membrane to generate the second mes- 
sengers, DAG and IP 3 . (b) Protein kinase C 
(PKC) is activated by DAG and Ca 2+ . 
Among the numerous effects of PKC is 
phosphorylation of 1 1< B , a cytoplasmic pro- 
tein that binds the transcription factor NF- 
kB and prevents it from entering the 
nucleus. Phosphorylation of IkB releases 
NF-kB, which then translocates into the 
nucleus, (c) Ca 2+ -dependent activation of 
calcineurin. Calcineurin is a Ca 2+ /calmod- 
ulin dependent phosphatase. IP 3 mediates 
the release of Ca 2+ from the endoplasmic 
reticulum. Ca 2+ binds the protein calmod- 
ulin, which then associates with and acti- 
vates the Ca 2+ /calmodulin-dependent 
phosphatase calcineurin. Active calcine- 
urin removes a phosphate group from 
NFAT, which allows this transcription fac- 



ERK), allows it to activate Elk, a transcription factor neces- 
sary for the expression of Fos. Phosphorylation of Fos by 
MAP kinase allows it to associate with Jun to form AP-1, 
which is an essential transcription factor for T-cell activation. 



Co-Stimulatory Signals Are Required 
for Full T-Cell Activation 



T-cell activation requires the dynamic 
membrane molecules described abov 
by itself, is not sufficient to fully 
T cells require more than one signal f( 
quent proliferation into effector cells: 



of multiple 



T cells. Naive 



Signal 1, the initial signal, is generated by interaction of 
an antigenic peptide with the TCR-CD3 complex. 



■ A subsequent antigen-nonspecific co-stimulatory signal, 
signal 2, is provided primarily by interactions between 
CD28 on the T cell and members of the B7 family on 
the APC. 

There are two related forms of B7, B7-1 and B7-2 (Figure 
10-13). These molecules are members of the immunoglobu- 
lin superfamily and have a similar organization of extracel- 
lular domains but markedly different cytosolic domains. 
Both B7 molecules are constitutively expressed on dendritic 
cells and induced on activated macrophages and activated B 
cells. The ligands for B7 are CD28 and CTLA-4 (also known 
as CD152), both of which are expressed on the T-cell mem- 
brane as disulfide-linked homodimers; like B7, they are 
members of the immunoglobulin superfamily (Figure 
10-13). Although CD28 and CTLA-4 are structurally similar 
glycoproteins, they act antagonistically. Signaling through 



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ofB-CellandT-Cell Respons 



TTTTT ¥ V ¥ V ¥ 

A L A A A Ras-GDP A A A A A 

•^(inactive) \ / GTP 

-GEFs 




s Ras-GDP V 


>> GDP 


(active) 
1 






MAP 


1 


kinase 


MAP kinase — . 


pathway 




xoooooooooc 






xoooooooooc 



n of the small C protein, Ras. Signals from 
theT-cell receptor result in activation of Ras via the action of specific 
guanine nucleotide exchange factors (GEFs) that catalyze the ex- 
change of GDP for GTP. Active Ras causes a cascade of reactions 
that result in the increased production of the transcription factor Fos. 
Following their phosphorylation, Fos and Jun dimerize to yield the 
transcription factor AP-1 . Note that all these pathways have impor- 
tant effects other than the specific examples shown in the figure. 

CD28 delivers a positive co-stimulatory signal to the T cell; 
signaling through CTLA-4 is inhibitory and down-regulates 
the activation of the T cell. CD28 is expressed by both resting 
and activated T cells, but CTLA-4 is virtually undetectable on 
resting cells. Typically, engagement of the TCR causes the in- 
duction of CTLA-4 expression, and CTLA-4 is readily de- 




Both B7 molecules are 
expressed on dendritic cells, 
activated macrophages, and 
activated B cells 



fl T H -cell activation requires 
provided by antigen-presenting cells (APCs) 
members on APCs with CD28 delivers the c 
gagement of the closely related CTLA-4 mo 
an inhibitory signal. All of these molecules contain at least one im- 
munoglobulin-like domain and thus belong to the immunoglobulin 
superfamily. [Adapted from P. S. Linsley and J. A. Ledbetter, 1993, 
Annu. Rev. Immunol. 11:191.] 



-nulatory signal. Er 
e with B7 produce 



tectable within 24 hours of stimulation, with maximal ex- 
pression within 2 or 3 days post-stimulation. Even though 
the peak surface levels of CTLA-4 are lower than those of 
CD28, it still competes favorably for B7 molecules because it 
has a significantly higher avidity for these molecules than 
CD28 does. Interestingly, the level of CTLA-4 expression is 
increased by CD28-generated co-stimulatory signals. This 
provides regulatory braking via CTLA-4 in proportion to the 
acceleration received from CD28. Some of the importance of 
CTLA-4 in the regulation of lymphocyte activation and pro- 
liferation is revealed by experiments with CTLA-4 knockout 
mice. T cells in these mice proliferate massively, which leads 
to lymphadenopathy (greatly enlarged lymph nodes), 
splenomegaly (enlarged spleen), and death at 3 to 4 weeks 
after birth. Clearly, the production of inhibitory signals by 
engagement of CTLA-4 is important in lymphocyte home- 
ostasis. 

CTLA-4 can effectively block CD28 co-stimulation by 
competitive inhibition at the B7 binding site, an ability that 
holds promise for clinical use in autoimmune diseases and 
transplantation. As shown in Figure 10-14, an ingeniously 
engineered chimeric molecule has been designed to explore 
the therapeutic potential of CTLA-4. The Fc portion of 
human IgG has been fused to the B7-binding domain of 
CTLA-4 to produce a chimeric molecule called CTLA-4Ig. 
The human Fc region endows the molecule with a longer 
half-life in the body and the presence of B7 binding domains 



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3 mac76:385 ,1 



^> </=* ] CTLA-4 binding domain 

Ys-sY 

IgGF c 



(b) B7 blockade by CTLA-4lg 




9 CTLA-4lg, a chimeric suppressor of co-stimulation. 

(a) CTLA-4lg, a genetically engineered molecule in which the Fc por- 
tion of human IgC is joined to the B7-binding domain of CTLA-4. 

(b) CTLA-4lg blocks costimulation by binding to B7 on antigen pre- 
senting cells and preventing the binding of CD28, a major co-stimula- 
tory molecule of T cells. 



allows it to bind to B7. A promising clinical trial of CTLA-4 
has been conducted in patients with psoriasis vulgaris, a 
T-cell-mediated autoimmune inflammatory skin disease. 
During the course of this trial, forty-three patients received 
four doses of CTLA-4Ig, and 46% of this group experienced 
a 50% or greater sustained improvement in their skin condi- 
tion. Further studies of the utility of CTLA-4Ig are also being 
pursued in other areas. 

Clonal Anergy Ensues If a Co-Stimulatory 
Signal Is Absent 

T H -cell recognition of an antigenic peptide-MHC complex 
sometimes results in a state of nonresponsiveness called 
clonal anergy, marked by the inability of cells to proliferate 
in response to a peptide-MHC complex. Whether clonal ex- 
pansion or clonal anergy ensues is determined by the pres- 
ence or absence of a co-stimulatory signal (signal 2), such as 
that produced by interaction of CD28 on T H cells with B7 on 
antigen-presenting cells. Experiments with cultured cells 
show that, if a resting T H cell receives the TCR-mediated sig- 
nal (signal 1) in the absence of a suitable co-stimulatory sig- 
nal, then the T H cell will become anergic. Specifically, if 
resting T H cells are incubated with glutaraldehyde-fixed 
APCs, which do not express B7 (Figure 10-15a), the fixed 
APCs are able to present peptides together with class II MHC 
molecules, thereby providing signal 1, but they are unable to 
provide the necessary co-stimulatory signal 2. In the absence 
of a co-stimulatory signal, there is minimal production of cy- 



tokines, especially of IL-2. Anergy can also be induced by in- 
cubating T H cells with normal APCs in the presence of the 
Fab portion of anti-CD28, which blocks the interaction of 
CD28 with B7 (Figure 10- 15b). 

Two different control experiments demonstrate that fixed 
APCs bearing appropriate peptide-MHC complexes can de- 
liver an effective signal mediated by T-cell receptors. In one 
experiment, T cells are incubated both with fixed APCs bear- 
ing peptide-MHC complexes recognized by the TCR of the T 
cells and with normal APCs, which express B7 (Figure 
10-15d). The fixed APCs engage the TCRs of the T cells, and 
the B7 molecules on the surface of the normal APCs cross- 
link the CD28 of the T cell. These T cells thus receive both 
signals and undergo activation. The addition of bivalent 
anti-CD28 to mixtures of fixed APCs and T cells also pro- 
vides effective co-stimulation by crosslinking CD28 (Figure 
10-15e). Well-controlled systems for studying anergy in 
vitro have stimulated considerable interest in this phenome- 
non. However, more work is needed to develop good animal 
systems for establishing anergy and studying its role in vivo. 

Superantigens Induce T-Cell Activation by 
Binding the TCR and MHC II Simultaneously 

Superantigens are viral or bacterial proteins that bind simul- 
taneously to the Vp domain of a T-cell receptor and to the a 
chain of a class II MHC molecule. Both exogenous and en- 
dogenous superantigens have been identified. Crosslinkage 
of a T-cell receptor and class II MHC molecule by either type 
of superantigen produces an activating signal that induces 
T-cell activation and proliferation (Figure 10-16). 

Exogenous superantigens are soluble proteins secreted by 
bacteria. Among them are a variety of exotoxins secreted by 
gram-positive bacteria, such as staphylococcal enterotoxins, 
toxic-shock-syndrome toxin, and exfoliative-dermatitis 
toxin. Each of these exogenous superantigens binds particu- 
lar Vp sequences in T-cell receptors (Table 10-3) and 
crosslinks the TCR to a class II MHC molecule. 

Endogenous superantigens are cell-membrane proteins 
encoded by certain viruses that infect mammalian cells. One 
group, encoded by mouse mammary tumor virus (MTV), 
can integrate into the DNA of certain inbred mouse strains; 
after integration, retroviral proteins are expressed on the 
membrane of the infected cells. These viral proteins, called 
minor lymphocyte stimulating (Mis) determinants, bind 
particular Vp sequences in T-cell receptors and crosslink the 
TCR to a class II MHC molecule. Four Mis superantigens, 
originating in different MTV strains, have been identified. 

Because superantigens bind outside of the TCR antigen- 
binding cleft, any T cell expressing a particular V p sequence 
will be activated by a corresponding superantigen. Hence, 
the activation is polyclonal and can affect a significant per- 
centage (5% is not unusual) of the total T H population. The 
massive activations that follow crosslinkage by a superanti- 
gen results in overproduction of T H -cell cytokines, leading 
to systemic toxicity. The food poisoning induced by staphy- 



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DfB-CellandT-Cell Respons 




1 Experimental demonstration of clonal anergy ve 
sus clonal expansion. (a,b) Only signal 1 is generated when restin 
T H cells are incubated with glutaraldehyde-fixed antigen-presentin 
cells (APCs) or with normal APCs in the presence of the Fab portio 



of 




iti-CD28. (c) The 
mal APCs. (d,e) In the pi 
CD28, both of which prodi 
activated by fixed APCs 



lococcal enterotoxins and the toxic shock induced by toxic- 
shock- syndrome toxin are two examples of the consequences 
of cytokine overproduction induced by superantigens. 

Superantigens can also influence T-cell maturation in the 
thymus. A superantigen present in the thymus during thymic 
processing will induce the negative selection of all thymo- 
cytes bearing a TCR V p domain corresponding to the super- 
antigen specificity. Such massive deletion can be caused by 
exogeneous or endogenous superantigens and is character- 
ized by the absence of all T cells whose receptors possess V p 
domains targeted by the superantigen. 



T-Cell Differentiation 

CD4 + and CD8 + T cells leave the thymus and enter the cir- 
culation as resting cells in the G stage of the cell cycle. There 
are about twice as many CD4 + T cells as CD8 + T cells in the 
periphery. T cells that have not yet encountered antigen 
(naive T cells) are characterized by condensed chromatin, 
very little cytoplasm, and little transcriptional activity. Naive 
T cells continually recirculate between the blood and lymph 



systems. During recirculation, naive T cells reside in sec- 
ondary lymphoid tissues such as lymph nodes. If a naive cell 
does not encounter antigen in a lymph node, it exits through 
the efferent lymphatics, ultimately draining into the thoracic 
duct and rejoining the blood. It is estimated that each naive T 
cell recirculates from the blood to the lymph nodes and back 
again every 12-24 hours. Because only about 1 in 10 5 naive T 
cells is specific for any given antigen, this large-scale recircu- 
lation increases the chances that a naive T cell will encounter 
appropriate antigen. 

Activated T Cells Generate Effector 
and Memory T Cells 

If a naive T cell recognizes an antigen-MHC complex on an 
appropriate antigen-presenting cell or target cell, it will be 
activated, initiating a primary response. About 48 hours after 
activation, the naive T cell enlarges into a blast cell and begins 
undergoing repeated rounds of cell division. As described 
earlier, activation depends on a signal induced by engage- 
ment of the TCR complex and a co-stimulatory signal in- 
duced by the CD28-B7 interaction (see Figure 10-13). These 
signals trigger entry of the T cell into the G! phase of the cell 



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superantigen 
membrane-bound 



Peptide for which 
TCR is not specific 

Class II MHC 



| Superantigen-mediated crosslinkage of T-cell re- 
ceptor and class II MHC molecules. A superantigen binds to allTCRs 
bearing a particular Vp sequence regardless of their antigenic speci- 
ficity. Exogenous superantigens are soluble secreted bacterial pro- 
teins, including various exotoxins. Endogenous superantigens are 
membrane-embedded proteins produced by certain viruses; they in- 
clude Mis antigens encoded by mouse mammary tumor virus. 



cycle and, at the same time, induce transcription of the gene 
for IL-2 and the a chain of the high-affinity IL-2 receptor. In 
addition, the co-stimulatory signal increases the half-life of 
the IL-2 mRNA. The increase in IL-2 transcription, together 
with stabilization of the IL-2 mRNA, increases IL-2 produc- 



tion by 100-fold in the activated T cell. Secretion of IL-2 and 
its subsequent binding to the high-affinity IL-2 receptor in- 
duces the activated naive T cell to proliferate and differenti- 
ate (Figure 10-17). T cells activated in this way divide 2-3 
times per day for 4-5 days, generating a large clone of prog- 
eny cells, which differentiate into memory or effector T-cell 
populations. 

The various effector T cells carry out specialized functions 
such as cytokine secretion and B-cell help (activated CD4 + T H 
cells) and cytotoxic killing activity (CD8 + CTLs). The genera- 
tion and activity of CTL cells are described in detail in Chapter 
14. Effector cells are derived from both naive and memory cells 
after antigen activation. Effector cells are short-lived cells, 
whose life spans range from a few days to a few weeks. The ef- 
fector and naive populations express different cell-membrane 
molecules, which contribute to different recirculation patterns. 

As described in more detail in Chapter 12, CD4 + effector 
T cells form two subpopulations distinguished by the differ- 
ent panels of cytokines they secrete. One population, called 
the T H 1 subset, secretes IL-2, IFN--y, and TNF-(3. The T H 1 
subset is responsible for classic cell-mediated functions, such 
as delayed-type hypersensitivity and the activation of cyto- 
toxic T lymphocytes. The other subset, called the T H 2 subset, 
secretes IL-4, IL-5, IL-6, and IL-10. This subset functions 
more effectively as a helper for B-cell activation. 

The memory T-cell population is derived from both naive 
T cells and from effector cells after they have encountered 
antigen. Memory T cells are antigen-generated, generally 



| TABLE 10-3^ 






Disease* 








V p SPECIFICITY 


Superantigen 


Mouse 


Human 


Staphylococcal enteroto 


ins 












SEA 




Food poiso 


ning 




1,3, 10, 11, 12, 17 


nd 


SEB 




Food poiso 


ning 




3,8.1,8.2,8.3 


3, 12, 14, 15, 17,20 


SEC1 




Food poiso 


ning 




7,8.2,8.3, 11 


12 


SEC2 




Food poiso 


ning 




8.2, 10 


12, 13, 14, 15, 17,20 


SEC3 




Food poiso 


ning 




7,8.2 


5, 12 


SED 




Food poiso 


ning 




3,7,8.3, 11, 17 


5, 12 


SEE 




Food poiso 


ning 




11, 15, 17 


5.1,6.1-6.3,8, 18 


Toxic-shock-syndrome tc 


xin (TSST1) 


Toxic-shod 






15, 16 


2 


Exfoliative-dermatitis to> 


in (ExFT) 


Scalded-sk 


nsynd 




10, 11, 15 


2 


Mycoplasma-arthritidis 
(MAS) 


upernatant 


Arthritis, si 


ock 




6,8.1-8.3 


nd 


Streptococcal pyrogenic 
(SPE-A, B, C, D) 


exotoxins 


Rheumatic 


fever, s 


hock 


nd 


nd 


'Disease results from infectic 


rbybaCteriatHatPr 


duce the indicated su 


e r ant, g e 


ns. 







A 



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ofB-CellandT-Cell Respons 




I Activation of a T H cell by both signal 1 and co- 
stimulatory signal 2 up-regulates expression of IL-2 and the high- 
affinity IL-2 receptor, leading to the entry of the T cell into the eel 
cycle and several rounds of proliferation. Some of the cells differenti- 
ate into effector cells, others into memory cells. 



long-lived, quiescent cells that respond with heightened reac- 
tivity to a subsequent challenge with the same antigen, gen- 
erating a secondary response. An expanded population of 
memory T cells appears to remain long after the population 
of effector T cells has declined. In general, memory T cells 
express many of the same cell-surface markers as effector T 
cells; no cell-surface markers definitively identify them as 
memory cells. 

Like naive T cells, most memory T cells are resting cells in 
the G stage of the cell cycle, but they appear to have less 
stringent requirements for activation than naive T cells do. 
For example, naive T H cells are activated only by dendritic 
cells, whereas memory T H cells can be activated by 
macrophages, dendritic cells, and B cells. It is thought that 
the expression of high levels of numerous adhesion mole- 
cules by memory T H cells enables these cells to adhere to a 
broad spectrum of antigen-presenting cells. Memory cells 
also display recirculation patterns that differ from those of 
naive or effector T cells. 



A CD4 + CD25 + Subpopulation of T cells 
Negatively Regulates Immune Responses 

Investigators first described T cell populations that could sup- 
press immune responses during the early 1970s. These cells 
were called suppressor T cells (T s ) and were believed to be 
CD8 + T cells. However, the cellular and molecular basis of the 
observed suppression remained obscure, and eventually great 
doubt was cast on the existence of CD8 + suppressor T cells. 
Recent research has shown that there are indeed T cells that 
suppress immune responses. Unexpectedly, these cells have 
turned out to be CD4 + rather than CD8 + T cells. Within the 
population of CD4 + CD25 + T cells, there are regulatory T cells 
that can inhibit the proliferation of other T cell populations in 
vitro. Animal studies show that members of the CD4 + CD25 + 
population inhibit the development of autoimmune diseases 
such as experimentally induced inflammatory bowel disease, 
experimental allergic encephalitis, and autoimmune diabetes. 
The suppression by these regulatory cells is antigen specific 
because it depends upon activation through the T cell recep- 
tor. Cell contact between the suppressing cells and their tar- 
gets is required. If the regulatory cells are activated by antigen 
but separated from their targets by a permeable barrier, no 
suppression occurs. The existence of regulatory T cells that 
specifically suppress immune responses has clinical implica- 
tions. The depletion or inhibition of regulatory T cells fol- 
lowed by immunization may enhance the immune responses 
to conventional vaccines. In this regard, some have suggested 
that elimination of T cells that suppress responses to tumor 
antigens may facilitate the development of anti-tumor immu- 
nity. Conversely, increasing the suppressive activity of regula- 
tory T cell populations could be useful in the treatment of 
allergic or autoimmune diseases. The ability to increase the 
activity of regulatory T cell populations might also be useful 
in suppressing organ and tissue rejection. Future work on this 
regulatory cell population will seek deeper insights into the 
mechanisms by which members of CD4 + CD25 + T cell popu- 
lations regulate immune responses. There will also be deter- 
mined efforts to discover ways in which the activities of these 
populations can be increased to diminish unwanted immune 
responses and decreased to promote desirable ones. 

Antigen-Presenting Cells Have Characteristic 
Co-Stimulatory Properties 

Only professional antigen-presenting cells (dendritic cells, 
macrophages, and B cells) are able to present antigen to- 
gether with class II MHC molecules and deliver the co-stim- 
ulatory signal necessary for complete T-cell activation that 
leads to proliferation and differentiation. The principal co- 
stimulatory molecules expressed on antigen-presenting cells 
are the glycoproteins B7-1 and B7-2 (see Figure 10-13). The 
professional antigen-presenting cells differ in their ability to 
display antigen and also differ in their ability to deliver the 
co-stimulatory signal (Figure 10-18). 



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Dendritic cell 




Macrophage 
Resting Activated 




Endocytosis 
phagocytosis 
(by Langerhans cells) 



Receptor-mediated 
endocytosis 



Class II MHC 



Constitutive B7 



Naive T cells 
Effector T cells 
Memory T cells 



I Differences in the properti 
itigen-presenting cells affect their ability 



Effector T cells 
Memory T cells 



if professional induce T-o 

present antigen and T cells doe 



Naive T cells 
Effector T cells 
Memory T cells 



of effector and memory 



Dendritic cells constitutively express high levels of class I 
and class II MHC molecules as well as high levels of B7-1 and 
B7-2. For this reason, dendritic cells are very potent activa- 
tors of naive, memory, and effector T cells. In contrast, all 
other professional APCs require activation for expression of 
co-stimulatory B7 molecules on their membranes; conse- 
quently, resting macrophages are not able to activate naive T 
cells and are poor activators of memory and effector T cells. 
Macrophages can be activated by phagocytosis of bacteria or 
by bacterial products such as LPS or by IFN-7, a T H 1 -derived 
cytokine. Activated macrophages up-regulate their expres- 
sion of class II MHC molecules and co-stimulatory B7 mole- 
cules. Thus, activated macrophages are common activators of 
memory and effector T cells, but their effectiveness in acti- 
vating naive T cells is considered minimal. 

B cells also serve as antigen-presenting cells in T-cell acti- 
vation. Resting B cells express class II MHC molecules but fail 
to express co-stimulatory B7 molecules. Consequently, rest- 
ing B cells cannot activate naive T cells, although they can ac- 
tivate the effector and memory T-cell populations. Upon 
activation, B cells up-regulate their expression of class II 
MHC molecules and begin expressing B7. These activated B 
cells can now activate naive T cells as well as the memory and 
effector populations. 



Cell Death and T-Cell Populations 

Cell death is an important feature of development in all 
multicellular organisms. During fetal life it is used to mold 
and sculpt, removing unnecessary cells to provide shape 
and form. It also is an important feature of lymphocyte 
homeostasis, returning T- and B-cell populations to their ap- 
propriate levels after bursts of antigen-induced proliferation. 
Apoptosis also plays a crucial role in the deletion of poten- 
tially autoreactive thymocytes during negative selection and 
in the removal of developing T cells unable to recognize self 
(failure to undergo positive selection). 

Although the induction of apoptosis involves different 
signals depending on the cell types involved, the actual death 
of the cell is a highly conserved process amongst vertebrates 
and invertebrates. For example, T cells may be induced to die 
by many different signals, including the withdrawal of 
growth factor, treatment with glucocorticoids, or TCR sig- 
naling. Each of these signals engages unique signaling path- 
ways, but in all cases, the actual execution of the cell involves 
the activation of a specialized set of proteases known as cas- 
pases. The role of these proteases was first revealed by studies 
of developmentally programmed cell deaths in the nematode 



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I Two pathways to apoptosis in T cells, (a) Acti- 
vated peripheral T cells are induced to express high levels of Fas and 
FasL. FasL induces the trimerization of Fas on a neighboring cell. 
FasL can also engage Fas on the same cell, resulting in a self- 
induced death signal. Trimerization of Fas leads to the recruitment 
of FADD, which leads in turn to the cleavage of associated mole- 
cules of procaspase 8 to form active caspase 8. Caspase 8 cleaves 
procaspase 3, producing active caspase 3, which results in the death 
of the cell. Caspase 8 can also cleave Bid to a truncated form that 
can activate the mitochondrial death pathway, (b) Other signals, 
such as the engagement of the TCR by peptide-MHC complexes on 
an APC, result in the activation of the mitochondrial death pathway. 
A key feature of this pathway is the release of AIF (apoptosis induc- 
ing factor) and cytochrome c from the inner mitochondrial mem- 
brane into the cytosol. Cytochrome c interacts with Apaf-1 and 
subsequently with procaspase 9 to form the active apoptosome. 
The apoptosome initiates the cleavage of procaspase 3, producing 
active caspase 3, which initiates the execution phase of apoptosis by 
proteolysis of substances whose cleavage commits the cell to apop- 
tosis. [Adapted in partfrom S. H. Kaufmann and M. O. Hengartner, 
2001. Trends Cell Biol. 7 7:526.] 



C. elegans, where the death of cells was shown to be totally 
dependent upon the activity of a gene that encoded a cysteine 
protease with specificity for aspartic acid residues. We now 
know that in mammals there are at least 14 cysteine proteases 
or caspases, and all cell deaths require the activity of at least a 
subset of these molecules. We also know that essentially every 
cell in the body produces caspase proteins, suggesting that 
every cell has the potential to initiate its own death. 

Cells protect themselves from apoptotic death under nor- 
mal circumstances by keeping caspases in an inactive form 
within a cell. Upon reception of the appropriate death signal, 
certain caspases are activated by proteolytic cleavage and then 
activate other caspases in turn, leading to the activation of effec- 
tor caspases. This catalytic cascade culminates in cell death. Al- 
though it is not well understood how caspase activation directly 
results in apoptotic death of the cell, presumably it is through 
the cleavage of critical targets necessary for cell survival. 

T cells use two different pathways to activate caspases 
(Figure 10-19). In peripheral T cells, antigen stimulation re- 
sults in proliferation of the stimulated T cell and production 
of several cytokines including IL-2. Upon activation, T cells 
increase the expression of two key cell-surface proteins in- 
volved in T-cell death, Fas and Fas ligand (FasL). When Fas 
binds its ligand, FasL, FADD (Fas-associated protein with 
death domain) is recruited and binds to Fas, followed by the 
recruitment of procaspase 8, an inactive form of caspase 8. 
The association of FADD with procaspase 8 results in the 
proteolytic cleavage of procaspase 8 to its active form; cas- 
pase 8 then initiates a proteolytic cascade that leads to the 
death of the cell. 

Outside of the thymus, most of the TCR-mediated apop- 
tosis of mature T cells is mediated by the Fas pathway. 
Repeated or persistent stimulation of peripheral T cells re- 
sults in the coexpression of both Fas and Fas ligand, followed 
by the apoptotic death of the cell. The Fas/FasL mediated 
death of T cells as a consequence of activation is called acti- 
vation-induced cell death (AICD) and is a major homeostatic 
mechanism, regulating the size of the pool of T cells and re- 
moving T cells that repeatedly encounter self antigens. 

The importance of Fas and FasL in the removal of acti- 
vated T cells is underscored by Ipr/lpr mice, a naturally occur- 
ring mutation that results in non-functional Fas. When T cells 
become activated in these mice, the Fas/FasL pathway is not 
operative; the T cells continue to proliferate, producing IL-2 
and maintaining an activated state. These mice spontaneously 
develop autoimmune disease, have excessive numbers of T 
cells, and clearly demonstrate the consequences of a failure to 
delete activated T cells. An additional mutation, gld/gld, is also 
informative. These mice lack functional FasL and display 
much the same abnormalities found in the Ipr/lpr mice. Re- 
cently, humans with defects in Fas have been reported. As 
expected, these individuals display characteristics of autoim- 
mune disease. (See the Clinical Focus box.) 

Fas and FasL are members of a family of related recep- 
tor/ligands including tumor necrosis factor (TNF) and its 



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ligand, TNFR (tumor necrosis factor receptor). Like Fas and 
FasL, membrane-bound TNFR interacts with TNF to induce 
apoptosis. Also similar to Fas/FasL-induced apoptosis, 
TNF/TNFR-induced death is the result of the activation of 
caspase 8 followed by the activation of effector caspases such 
as caspase 3. 

In addition to the activation of apoptosis through death 
receptor proteins like Fas and TNFR, T cells can die through 
other pathways that do not activate procaspase 8. For exam- 
ple, negative selection in the thymus induces the apoptotic 
death of developing T cells via a signaling pathway that orig- 
inates at the TCR. We still do not completely understand why 
some signals through the TCR induce positive selection and 
others induce negative selection, but we know that the 
strength of the signal plays a critical role. A strong, negatively 
selecting signal induces a route to apoptosis in which 
mitochondria play a central role. In mitochondrially depen- 
dent apoptotic pathways, cytochrome c, which normally 
resides in the inner mitochondrial membrane, leaks into the 
cytosol. Cytochrome c binds to a protein known as Apaf-1 
(apoptotic protease-activating factor- 1) and undergoes an 
ATP-dependent conformational change and oligomeriza- 
tion. Binding of the oligomeric form of Apaf-1 to procaspase 
9 results in its transformation to active caspase 9. The com- 
plex of cytochrome c/ Apaf-1 /caspase 9, called the apopto- 
some, proteolytically cleaves procaspase 3 generating active 
caspase 3, which initiates a cascade of reactions that kills the 
cell (Figure 10-19). Finally, mitochondria also release an- 
other molecule, AIF (apoptosis inducing factor), which plays 
a role in the induction of cell death. 

Cell death induced by Fas/FasL is swift, with rapid activa- 
tion of the caspase cascade leading to cell death in 2-4 hours. 
On the other hand, TCR-induced negative selection appears 
to be a more circuitous process, requiring the activation of sev- 
eral processes including mitochondrial membrane failure, the 
release of cytochrome c, and the formation of the apopto- 
some before caspases become involved. Consequently, TCR- 
mediated negative selection can take as long as 8-10 hours. 

An important feature in the mitochondrially induced cell 
death pathway is the regulatory role played by Bcl-2 family 
members. Bcl-2 and Bcl-XL both reside in the mitochondr- 
ial membrane. These proteins are strong inhibitors of apop- 
tosis, and while it is not clear how they inhibit cell death, one 
hypothesis is that they somehow regulate the release of cy- 
tochrome c from the mitochondria. There are at least three 
groups of Bcl-2 family members. Group I members are anti- 
apoptotic and include Bcl-2 and Bcl-xL. Group II and Group 
III members are pro-apoptotic and include Bax and Bak in 
Group II and Bid and Bim in Group III. There is clear evi- 
dence that levels of anti-apoptotic Bcl-2 family members play 
an important role in regulating apoptosis in lymphocytes. 
Bcl-2 family members dimerize, and the anti-apoptotic 
group members may control apoptosis by dimerizing with 
pro-apoptotic members, blocking their activity. As indicated 
in Figure 10-19, cleavage of Bid, catalyzed by caspase 8 gen- 



erated by the Fas pathway, can turn on the mitochondrial 
pathway. Thus signals initiated through Fas can also involve 
the mitochondrial death pathway. 

While it is apparent there are several ways a lymphocyte 
can be signaled to die, all of these pathways to cell death con- 
verge upon the activation of caspases. This part of the cell- 
death pathway, the execution phase, is common to almost all 
death pathways known in both vertebrates and invertebrates, 
demonstrating that apoptosis is an ancient process that has 
been conserved throughout evolution. 



Peripheral 78 T Cells 



In 1986, a small population of peripheral-blood T cells was 
discovered that expressed CD3 but failed to stain with mon- 
oclonal antibody specific for the a(3 T-cell receptor, indicat- 
ing an absence of the a (3 heterodimer. Many of these cells 
eventually were found to express the 78 receptor. 

gd T Cells Are Far Less Pervasive 
Than abT Cells 

In humans, less than 5% of T cells bear the 78 heterodimer, 
and the percentage of 78 T cells in the lymphoid organs of 
mice has been reported to range from 1% to 3%. In addition 
to their presence in blood and lymphoid tissues, they also ap- 
pear in the skin, intestinal epithelium, and pulmonary epithe- 
lium. Up to 1% of the epidermal cells in the skin of mice are 
78 T cells. In general, 78 T cells are not MHC-restricted, and 
most do not express the coreceptors CD4 and CD8 present on 
populations of a (3 T cells. Although the potential of the 7 and 
8 TCR loci to generate diversity is great, very little diversity is 
found in this type of T cell. In fact, as pointed out in Chapter 
9, most of the 78 T cells in humans have an identical combi- 
nation of 78 chains (79 and 82). 

78 T Cells Recognize Nonpeptide Ligands 

Not all T cells are self-MHC restricted and recognize only 
peptide antigens displayed in the cleft of the self-MHC mol- 
ecule. Indeed, Chapters 2 and 8 describe a(3 TCR-bearing T 
cells (NK1-T cells and CD 1- restricted T cells) that are not re- 
stricted by conventional MHC molecules. In one study, a 78 
T-cell clone was found to bind directly to a herpes-virus pro- 
tein without requiring antigen processing and presentation 
together with MHC. Human 78 T cells have been reported 
that display MHC-independent binding of a phospholipid 
derived from M. tuberculosis, the organism responsible for 
tuberculosis (see Chapter 9). This finding suggests that in 
many cases the TCR receptors of 78 T cells bind to epitopes 
in much the same way that the immunoglobulin receptors 
of B cells do. The fact that most human 78 T cells all have 
the same specificity suggests that like other components of 
e system, they recognize and respond to 



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ofB-CeilandT-Cell Respons 



CLINICAL FOCUS 



Patient C: An 8-year-old boy, the son of 



Failure of Apoptosis Causes 
Defective Lymphocyte 
Homeostasis 



Th 



maintenance of appropriate 
numbers of various types of lymphocytes 
is extremely important to an effective 
immune system. One of the most im- 
portant elements in this regulation is 
apoptosis mediated by the Fas/FasL 
ligand system. The following excerpts 
from medical histories show what can 
happen when this key regulatory mecha- 
nism fails. 

Patient A: A woman, now 43, has had a 
long history of immunologic imbalances 
and other medical problems. By age 2, 
she was diagnosed with the Canale- 
Smith syndrome (CSS), a severe enlarge- 
ment of such lymphoid tissues as lymph 
nodes (lymphadenopathy) and spleen 
(splenomegaly). Biopsy of lymph nodes 
showed that, in common with many 
other CSS patients, she had greatly in- 
creased numbers of lymphocytes. She 
had reduced numbers of platelets 
(thrombocytopenia) and, because her 
red blood cells were being lysed, she was 
anemic (hemolytic anemia). The reduc- 
tion in numbers of platelets and the lysis 
of red blood cells could be traced to the 
action of circulating antibodies that re- 
acted with these host components. At 
age 21, she was diagnosed with grossly 
enlarged pelvic lymph nodes that had to 
be removed. Ten years later, she was 
again found to have an enlarged abdom- 
inal mass, which on surgical removal 
turned out to be a half-pound lymph- 
node aggregate. She has continued to 
have mild lymphadenopathy and, typical 
of these patients, the lymphocyte popu- 
lations of enlarged nodes had elevated 
numbers of T cells (87% as opposed to a 
normal range of 48%-67% T cells). Ex- 



n of thes 



s by flow cytome- 
try and fluorescent antibody staining re- 
vealed an excess of double-negative T 
cells (see illustration below). Also, like 
many patients with Canale-Smith syn- 
drome, she has had cancer, breast can- 
cer at age 22 and skin cancer at ages 22 
and 41. 

Patient B: A man who was eventually di- 
agnosed with Canale-Smith syndrome 
had severe lymphadenopathy and spleno- 
megaly as an infant and child. He was 
treated from age 4 to age 12 with corti- 
costeroids and the immunosuppressive 
drug mercaptopurine. These appeared 
to help, and the swelling of lymphoid tis- 
sues became milder during adolescence 
and adulthood. At age 42, he died of liver 



Normal control 



patient B, was z 


Iso afflicted with Canale- 


Smith syndrom 


s and showed elevated T- 


cell counts and 


severe lymphadenopathy 


at the age of se 


/en months. At age 2 his 


spleen became 


so enlarged that it had to 


be removed. 


He also developed he- 


molytic anemia 


and thrombocytopenia. 


However, altho 


gh he continued to have 


elevated T-cell c 


ounts, the severity of his 


hemolytic anen 


lia and thrombocytope- 



nia have so far been controlled by treat- 
ment with methotrexate, a DNA- synthe- 
sis-inhibiting drug used for immunosup- 
pression and cancer chemotherapy. 

Recognition of the serious conse- 
quences of a failure to regulate the num- 
ber of lymphocytes, as exemplified by 
these case histories, emerged from de- 
tailed study of several children whose 
enlarged lymphoid tissues attracted 
medical attention. In each of these cases 
of Canale-Smith syndrome, examination 
revealed grossly enlarged lymph nodes 
that were 1-2 cm in girth and some- 
times large enough to distort the local 
anatomy. In four of a group of five chil- 
dren who were studied intensively, the 





Patient A 






= 24% 






1".. 


I CD47CD8+ 




r 




r /■'•'; ;. 








- 43% 






32% 


' CD47CD8" 




CD4+ 


/CDS" 


= , 


, 







CD4 CD4 

Flow-cytometric analysis of T cells in the blood of Patient A and a control subject. Th 
relative staining by an anti-CD8 antibody is shown on the y axis and the relative stain 
by an anti-CD4 antibody appears on the x axis. Mature T cells are either CD4 + or 
CD8 + . While almost all of the T cells in the control subject are CD4 + or CD8 + , the C 
patient shows high numbers of double-negative T cells (43%), which express neither 
CD4 nor CD8. The percentage of each category of T cells is indicated in the quadrant 
[Adapted from Drappa et al., 1996, New England Journal of Medicine 335:1643.] 



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J Normal controls 
I Patient A 
| Patient B 



1 



Anti-Fas antibo 

Fas-mediated killing takes place when Fas is crosslinked by FasL, its normal 1 
by treatment with anti-Fas antibody, which artificially crosslinks Fas molecule: 
experiment shows the reduction in numbers of T cells after induction of apo| 
cells from patients and controls by crosslinking Fas with increasing amounts 
Fas monoclonal antibody. T cells from the Canale-Smith patients (A and B) a 
to Fas-mediated death. [Adapted from Drappa et al., 1996, New England Jou 
Medicine 335:1643.] 



spleens were so massive that they had to 
be removed. 

Even though the clinical picture in 
Canale-Smith syndrome can vary from 
person to person, with some individuals 
suffering severe chronic affliction and 
others only sporadic episodes of illness, 
there is a common feature, a failure of 
activated lymphocytes to undergo Fas- 
mediated apoptosis. Isolation and se- 
quencing of Fas genes from a number of 
patients and more than 100 controls re- 
veals that CSS patients are heterozygous 
{fas +/ ~) at the fas locus and thus carry 
one copy of a defective fas gene. A com- 
parison of Fas-mediated cell death in T 
cells from normal controls who do not 
carry mutant Fas genes with death in- 
duced in T cells from CSS patients, 
shows a marked defect in Fas-induced 
death (see illustration above). Character- 
ization of the Fas genes so far seen in 
CSS patients reveals that they have mu- 
tations in or around the region encoding 
the death-inducing domain (the "death 
domain") of this protein (see illustration 



below). Such mutations result in the pro- 
duction of Fas protein that lacks biologi- 
cal activity but still competes with 
normal Fas molecules for interactions 
with essential components of the Fas- 
mediated death pathway. Other muta- 
tions have been found in the extracellular 
domain of Fas, often associated with 
milder forms of CSS or no disease at all. 
A number of research groups have 
conducted detailed clinical studies of 
CSS patients, and the following general 
observations have been made: 



■ The cell populations of the blood and 
lymphoid tissues of CSS patients 
show dramatic elevations (5-fold to as 
much as 20-fold) in the numbers of 
lymphocytes of all sorts, including T 
cells, B cells, and NK cells. 
Most of the patients have elevated 
levels of one or more classes of im- 
munoglobulin (hyper-gammaglobulin- 

Immune hyperactivity is responsible 
for such autoimmune phenomena 
as the production of autoantibodies 
against red blood cells, resulting in he- 
molytic anemia, and a depression in 
platelet counts due to the activity of 
anti-platelet auto-antibodies. 

These observations establish the impor- 
tance of the death-mediated regulation 
of lymphocyte populations in lympho- 
cyte homeostasis. Such death is neces- 
sary because the immune response to 
antigen results in a sudden and dramatic 
increase in the populations of respond- 
ing clones of lymphocytes and temporar- 
ily distorts the representation of these 
clones in the repertoire. In the absence 
of cell death, the periodic stimulation of 
lymphocytes that occurs in the normal 
course of life would result in progres- 
sively increasing, and ultimately unsus- 
tainable, lymphocyte levels. As the 
Canale-Smith syndrome demonstrates, 
without the essential culling of lympho- 
cytes by apoptosis, severe and life-threat- 
ening disease can result. 







. The fas gene is composed of 9 exons separated by 8 introns. Exons 
extracellular part of the protein, exon 6 encodes the transmembrane 
region, and exons 7-9 encode the intracellular region of the molecule. Much of exon 9 
is responsible for encoding the critical death domain. [Adapted from C. H. Fisher et al., 
1995, Cell 81:935.] 



Map of fas 
1-5 encode the 



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8/29/02 10:23 AM Page 24< 



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ofB-CeilandT-Cell Respons 



molecular patterns that are found in certain pathogens but 
not in humans. Thus they may play a role as first lines of de- 
fense against certain pathogens, expressing effector functions 
that help control infection and secreting cytokines that pro- 
mote an adaptive immune response by a(3 T cells and B cells. 



SUMMARY 

■ Progenitor T cells from the bone marrow enter the thymus 
and rearrange their TCR genes. In most cases these thymo- 
cytes rearrange a(5 TCR genes and become a3 T cells. A small 
minority rearrange 78 TCR genes and become 78 T cells. 

■ The earliest thymocytes lack detectable CD4 and CD8 and 
are referred to as double-negative cells. During develop- 
ment, the majority of double-negative thymocytes develop 
into CD4 + CD8~ a0 T cells or CD4~CD8 + ap T cells. 

■ Positive selection in the thymus eliminates T cells unable 
to recognize self-MHC and is the basis of MHC restriction. 
Negative selection eliminates thymocytes bearing high- 
affinity receptors for self-MHC molecules alone or self- 
antigen plus self-MHC and produces self-tolerance. 

■ T H -cell activation is initiated by interaction of the TCR- 
CD3 complex with a peptide-MHC complex on an anti- 
gen-presenting cell. Activation also requires the activity of 
accessory molecules, including the coreceptors CD4 and 
CD8. Many different intracellular signal-transduction 
pathways are activated by the engagement of the TCR. 

■ T-cells that express CD4 recognize antigen combined with 
a class II MHC molecule and generally function as T H cells; 
T cells that express CD8 recognize antigen combined with 
a class I MHC molecule and generally function as T c cells. 

■ In addition to the signals mediated by the T-cell receptor 
and its associated accessory molecules (signal 1), activa- 
tion of the T H cell requires a co-stimulatory signal (signal 
2) provided by the antigen-presenting cell. The co-stimu- 
latory signal is commonly induced by interaction between 
molecules of the B7 family on the membrane of the APC 
with CD28 on the T H cell. Engagement of CTLA-4, a close 
relative of CD28, by B7 inhibits T-cell activation. 

■ TCR engagement with antigenic peptide-MHC may in- 
duce activation or clonal anergy. The presence or absence 
of the co-stimulatory signal (signal 2) determines whether 
activation results in clonal expansion or clonal anergy. 

■ Naive T cells are resting cells (G ) that have not encountered 
antigen. Activation of naive cells leads to the generation of ef- 
fector and memory T cells. Memory T cells, which are more 
easily activated than naive cells, are responsible for secondary 
responses. Effector cells are short lived and perform helper, 
cytotoxic, or delayed-type hypersensitivity functions. 

■ The T-cell repertoire is shaped by apoptosis in the thymus 
and periphery. 

■ 78 T cells are not MHC restricted. Most in humans bind 
free antigen, and most have the same specificity. They may 
function as part of the innate immune system. 



References 

Ashton-Rickardt, P. G., A. Bandeira, J. R. Delaney, L. Van Kaer, 
H. P. Pircher, R. M. Zinkernagel, and S. Tonegawa. 1994. 
Evidence for a differential avidity model of T-cell selection in 
the thymus. Cell 74:577. 

Drappa, M. D., A. K. Vaishnaw, K. E. Sullivan, B. S. Chu, and 
K. B. Elkon. 1996. Fas gene mutations in the Canale-Smith 
syndrome, an inherited lymphoproliferative disorder associ- 
ated with autoimmunity. New England Journal of Medicine 
335:1643. 

Dutton, R. W., L. M. Bradley, and S. L. Swain. 1998. T-cell mem- 
ory. Annu. Rev. Immunol. 16:201. 

Ellmeier, W., S. Sawada, and D. R. Littman. 1999. The regulation 
of CD4 and CD8 coreceptor gene expression during T-cell de- 
velopment. Annu. Rev. Immunol. 17:523. 

Hayday, A. 2000. 78 Cells: A right time and right place for a con- 
served third way of protection. Annu. Rev. Immunol. 18:1975. 

Herman, A, J. W. Kappler, P. Marrack, and A. M. Pullen. 1991. 
Superantigens: mechanism of T-cell stimulation and role in 
immune responses. Annu. Rev. Immunol. 9:745. 

Lanzavecchia, A., G. Lezzi, and A. Viola. 1999. From TCR en- 
gagement to T-cell activation: a kinetic view of T-cell behavior. 
Cell 96:1. 

Myung, P. S., N. I. Boerthe, and G. A. Koretzky. 2000. Adapter 
proteins in lymphocyte antigen-receptor signaling. Curr. Opin. 
Immunol. 12:256. 



of homeostasis 



Osborne, B. A. 1996. Apoptosis 
in the immune system. Curr. Opin. Immunol. 8:245. 

Osborne, B., A. 2000. Transcriptional control of T-cell develop- 
ment. Curr. Opin. Immunol. 12:301. 

Owen, J. J. T, and N. C. Moore. 1995. Thymocyte-stromal-cell 
interactions and T-cell selection. Immunol. Today 16:336. 

Salomon, B., and I. A. Bluestone. 2001. Complexities of 
CD28/B7: CTLA-4 costimulatory pathways in autoimmunity 
and transplantation. Annu. Rev. Immunol. 19:225. 

Schreiber, S. L., and G R. Crabtree. 1992. The mechanism of ac- 
tion of cyclosporin A and FK506. Immunol. Today 13:136. 

Thompson, C. B. and J. C. Rathmell. 1999. The central effectors 
of cell death in the immune system. Annu. Rev. Immunol. 
17:781. 

Vaishnaw, A. K., J. R. Orlinick, J. L. Chu, P. H. Krammer, M. V 
Chao, and K. B. Elkon. 1999. The molecular basis for apoptotic 
defects in patients with CD95 (Fas/Apo-1) mutations. Journal 
of Clinical Investigation 103:355. 



USEFUL WEB SITES 

http://www.ncbi.nlm.nih.gov/Omim/ 
http://www.ncbi.nlm.nih.gov/htbinpost/Omim/ge 

The Online Mendelian Inheritance in Man Web si 
a subsite that features ten different inherited diseases associ- 
ated with defects in the TCR complex or associated proteins. 



A 



10:23 AM Page 245 n 



'^ 



http://www.ultranet.eom/~jkimball/BiologyPages/A/ 

Apoptosis.html 
http://www.ultranet.eom/~jkimball/BiologyPages/B/ 

B_and_Tcells.html 

These subsites of John Kimball's Biology Pages Web site pr< 
vide a clear introduction to T-cell biology and a good bas 
discussion of apoptosis. 



http://w 



e.org/knockout/kn< 



e.htm 



Within the Frontiers in Bioscience Database of Gene Knock- 
outs, one can find information on the effects of knockouts of 
many genes involved in the development and function of cells 
of the T cells. 



of T H cells leads to the release or induc- 
nuclear factors that activate gene transcrip- 



factors that support proliferation of 
present in the cytoplasm of resting 



a. What trarj 
activated T H cells 
T H cells in inactive forms? 

b. Once in the nucleus, what might these transcription fac- 
tors do? 

k You have fluorescein-labeled anti-CD4 and rhodamine- 
labeled anti-CD8. You use these antibodies to stain thymo- 
cytes and lymph-node cells from normal mice and from 
RAG-1 knockout mice. In the diagrams below, draw the 
FACS plots that you would expect. 



Study Questions 

Clinical Focus Question Over a period of several years, a 
group of children and adolescents are regularly dosed with com- 
pound X, a life-saving drug. However, in addition to its beneficial 
effects, this drug interferes with Fas-mediated signaling. 

a. What clinical manifestations of this side effect of com- 
pound X might be seen in these patients? 

b. If white blood cells from an affected patient are stained 
with a fluorescein-labeled anti-CD4 and a phycoerythrin- 
labeled anti-CD8 antibody, what might be seen in the 
flow-cytometric analysis of these cells? What pattern 
would be expected if the same procedure were performed 
on white blood cells from a healthy control? 

1 . You have a CD8 + CTL clone (from an H-2* mouse) that has 
a T-cell receptor specific for the H-Y antigen. You clone the 
ap TCR genes from this cloned cell line and use them to pre- 
pare transgenic mice with the H-2 1 or H-2 rf haplotype. 

a. How can you distinguish the immature thymocytes from 
the mature CD8 + thymocytes in the transgenic mice? 

b. For each transgenic mouse listed in the table below, indi- 
cate with ( + ) or ( - ) whether the mouse would have im- 
mature double-positive and mature CD8 + thymocytes 
bearing the transgenic T-cell receptor. 



Transgenic 
mouse 


Immature 
thymocytes 


Mature CD8 + 
thymocytes 


H-2 female 






H-2* male 






H-2 d female 






H-2 d male 







c. Explai 

d. Explai 



for the H-2 transgenics, 
for the H-2 transgenics. 



2. Cyclosporin and FK506 are powerful immunosuppressive 
drugs given to transplant recipients. Both drugs prevent the 
on of a complex between calcineurin and 
Ca 2+ /calmodulin. Explain why these compounds suppress T- 
cell-mediated aspects of transplant rejection. Hint: see Figure 
10-11. 




RAG-1 knockout rr 



Lymph node 
Normal mice RAG-1 kr 



5. In order to demonstrate positive thymic selection experi- 

', researchers analyzed the thymocytes from normal 
H-2 6 mice, which have a deletion of the class II IE gene, and 
from H-2 fc mice in which the class II LA gene had been 
knocked out. 

a. What MHC molecules would you find on antigen-pre- 
senting cells from the normal H-2 fc mice? 

b. What MHC molecules would you find on antigen-pre- 
senting cells from the IA knockout H-2'' mice? 

c. Would you expect to find CD4 + T cells, CD8 + T cells, or 
both in each type of mouse? Why? 

6. In his classic chimeric-mouse experiments, Zinkernagel 
took bone marrow from mouse 1 and a thymus from mouse 
2 and transplanted them into mouse 3, which was thymec- 
tomized and lethally irradiated. He then challenged the re- 
constituted mouse with LCM virus and removed its spleen 
cells. These spleen cells were then incubated with LCM-in- 
fected target cells with different MHC haplotypes, and the 
lysis of the target cells was monitored. The results of two 



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8/29/02 2:31 PM Page 246 



ofB-CellandT-Cell Respons 



Experiment 


Bone-marrow donor 


Thymectomized, 
x-irradiated recipient 


Lysis of LCM -infected target cells 


H-2'' H-2 1 H-2 b 


A 


C57BL/6 X BALB/c 


C57BL/6 X BALB/c 


+ - - 


B 


C57BL/6 X BALB/c 


C57BL/6 X BALB/c 


- - + 



such experiments using H-2 ' strain C57BL/6 mice and H-2 
strain BALB/c mice are shown in the table on the above. 

a. What was the haplotype of the thymus-donor strain in 
experiment A and experiment B? 

b. Why were the H-2 target cells not lysed in experiment A 
but were lysed in experiment B? 

c. Why were the H-2 target cells not lysed in either experi- 



. You wish to determine the percentage of various types of 
thymocytes in a sample of cells from mouse thymus using 
the indirect immunofluorescence method. 



7. Fill in the blank(s) in each statement below (a 
most appropriate term(s) from the following lis 
be used once, more than once, or not at all. 



-k) with the 
. Terms may 



protein phosphatase(s) 


CD8 


Class 1 MHC 


CD45 


protein kinase(s) 


CD4 


Class II MHC 


B7 


CD28 


IL-2 


IL-6 


CTLA-4 



a. Lck and ZAP- 70 are . 

b. is a T-cell membrane protein that has cytosolic 

domains with phosphatase activity. 

c. Dendritic cells express constitutively, whereas B 

cells must be activated before they express this mem- 
brane molecule. 

d. Calcineurin, a , is involved in generating the ac- 
tive form of the transcription factor NFAT. 

e. Activation of T H cells results in secretion of and 

expression of its receptor, leading to proliferation and 
differentiation. 

f. The co-stimulatory signal needed for complete T H -cell 

activation is triggered by interaction of on the T 

cell and on the APC. 

g. Knockout mice lacking class I MHC molecules fail to 
produce thymocytes bearing . 

h. Macrophages must be activated before they express 

molecules and molecules. 

i. T cells bearing are absent from the lymph nodes 

of knockout mice lacking class II MHC molecules. 

j. PIP 2 is split by a to yield DAG and IP 3 . 

k. In activated T H cells, DAG activates a , which acts 

to generate the transcription factor NF-kB. 
I. stimulates and inhibits T-cell activation 

when engaged by or on antigen-presenting cells. 



a. You first stain the sample with goat anti-CD3 (primary 
antibody) and then with rabbit FITC-labeled anti-goat Ig 
(secondary antibody), which emits a green color. Analysis 
of the stained sample by flow cytometry indicates that 
70% of the cells are stained. Based on this result, how 
many of the thymus cells in your sample are expressing 
antigen-binding receptors on their surface? Would all be 
expressing the same type of receptor? Explain your an- 
swer. What are the remaining unstained cells likely to be? 

b. You then separate the CD3 + cells with the fluorescence- 
activated cell sorter (FACS) and restain them. In this case, 
the primary antibody is hamster anti-CD4 and the sec- 
ondary antibody is rabbit PE-labeled anti-hamster-Ig, 
which emits a red color. Analysis of the stained CD3 + 
cells shows that 80% of them are stained. From this re- 
sult, can you determine how many T c cells are present in 
this sample? If yes, then how many T c cells are there? If 
no, what additional experiment would you perform in 
order to determine the number of T c cells that are pre- 



9. Many of the effects of engaging the TCR with MHC-peptide 
can be duplicated by the administration of ionomycin plus a 
phorbol ester. Ionomycin is a Ca 2+ ionophore, a compound 
that allows calcium ions in the medium to cross the plasma 
membrane and enter the cell. Phorbol esters are analogues of 
diacylglycerol (DAG). Why does the administration of phor- 
bol and calcium ionophores mimic many effects of TCR en- 
gagement? 

1 0. What effects on cell death would you expect to observe in 
mice carrying the following genetic modifications? Justify 



you 

a. Mice that are transgenic for BCL-2 and over-express this 

b. Mice in which caspase 8 has been knocked out. 

c. Mice in which caspase 3 has been knocked out. 

1 1 . Several basic themes of signal transduction were identified 
and discussed in this chapter. What are these themes? Con- 
sider the signal-transduction processes of T-cell activation 
and provide an example for each of six of the seven themes 



A 



8536d_chlO_221-24 r 



3:58 PM Page 24' 



:76 mac76:385 ,1 



-A 



B-Cell Generation, 
Activation, and 
Differentiation 



THE DEVELOPMENTAL PROCESS THAT RESULTS IN 
production of plasma cells and memory B cells 
can bedivided into three broad stages: generation 
of mature, immunocompetent B cells (maturation), activa- 
tion of mature B cells when they interact with antigen, and 
differentiation of activated B cells into plasma cells and 
memory B cells. In many vertebrates, including humans 
and mice, the bone marrow generates B cells. This process 
is an orderly sequence of Ig-gene rearrangements, which 
progresses in the absence of antigen. This is the antigen- 
independent phase of B-cell development. 

A mature B cell leavesthe bone marrow expressing mem- 
brane-bound immunoglobulin (mlgM and mlgD) with a 
single antigenic specificity. These naive B cells, which have 
not encountered antigen, circulate in the blood and lymph 
and are carried to thesecondary lymphoid organs, most no- 
tably the spleen and lymph nodes (see Chapter 2). If a B cell 
is activated by the antigen specific to its membrane-bound 
antibody, the eel I proliferates(clonal expansion) and differen- 
tiates to generate a population of antibody- secreting plasma 
cells and memory B cells. In this activation stage, affinity 
maturation istheprogressiveincreasein theaverage affinity 
of theanti bodies produced and class switching isthechange 
in theisotypeof theanti body produced by the B eel I from n 
to 7, a, ore. Since B cell activation and differentiation in the 
periphery require antigen, this stage comprises the antigen- 
dependent phase of B-cell development. 

M any B cells are produced in the bone marrow through- 
out life, but very few of thesecells mature. In mice, thesizeof 
the recirculating pool of B cells is about 2 x 10 8 cells. M ost 
of these eel Is circulate as naive B cells, which have short life 
spans (half lives of less than 3 days to about 8 weeks) if they 
fail to encounter antigen or lose in the competition with 
other Bcellsforresidencein a supportivelymphoid environ- 
ment. Given that theimmunesystem is able to generate a to- 
tal antibody diversity that exceeds 10 9 , clearly only a small 
fraction of this potential repertoire is displayed at any time 
by membrane immunoglobulin on recirculating B cells. In- 
deed, throughout the life span of an animal, only a small 
fraction of the possible antibody diversity is ever generated. 



chapt 







; B-Cell Maturation 

-, B-Cell Activation and Proliferation 

; The Humoral Response 

-, In Vivo Sites for Induction of Humoral Responses 

; Germinal Centers and Antigen-Induced B-Cell 
Differentiation 

; Regulation of B-Cell Development 

; Regulation of the Immune Effector Response 



Some aspects of B-cell developmental processes have 
been described in previous chapters. The overall pathway, 
beginning with the earliest distinctive B-lineage cell, is de- 
scribed in sequence in this chapter. Figure 11-1 presents an 
overview of the major events in humans and mice. M ost of 
this chapter applies to humans and mice, but important 
departures from these developmental pathways have been 
shown to occur in some other vertebrates. Finally, this chap- 
ter will consider the regulation of B-cell development at var- 
ious stages. 



B-Cell Maturation 

The generation of mature B cells first occurs in the embryo 
and continues throughout life. Before birth, the yolk sac, 
fetal liver, and fetal bonemarrowarethemajorsitesof B-cell 
maturation; after birth, generation of mature B cells occurs 
in the bone marrow. 



248 



Generation of B-Cell and T-Cell Responses 



VI SUALIZI N G CONCEPTS 



ANTIGEN-INDEPENDENT PHASE 
(maturation) 





P) 


rearrangement 


CD45R / 


Selection 


(B220) L- 






surfaceV 
marker 




Bone marrow 



ANTIGEN -DEPENDENT PHASE 
(activation and differentiation) 




Peripheral lymphoid organ 



■J[mid*»Bl Overview of B-cel I development. During the anti- 
gen-independent maturation phase immunocompetent B cells 
expressing membrane IgM and IgD are generated in the bone 
marrow. Only about 10% of the potential B cells reach maturity 
and exit the bone marrow. N aive B cells in the periphery die within 
a few days unless they encounter soluble protein antigen and ac- 



tivated T H cells. Once activated, B cells proliferate within sec- 
ondary lymphoid organs. Those bearing high-affinity mlg differ- 
entiate into plasma cells and memory B cells, which may express 
different isotypes because of class switching. The numbers cited 
refer to B-cell development in the mouse, but the overall princi- 
ples applyto humans as well. 



Progenitor B Cells Proliferate 
in Bone Marrow 

B-cell development begins as lymphoid stem eel Is differenti- 
ate into the earliest distinctive B-lineage cell— the progeni- 
tor B cell (pro-B cell)— which expresses a transmembrane 
tyrosine phosphatase called CD45R (sometimes called B220 
in mice). Pro-B cells proliferate within the bone marrow, fill- 
ing the extravascular spaces between large sinusoids in the 



shaft of a bone. Proliferation and differentiation of pro-B 
cells into precursor B cells (pre-B cells) requires the micro- 
environment provided by the bone-marrow stromal cells. If 
pro-B cells are removed from the bone marrow and cultured 
in vitro, they will not progress to more mature B-cell stages 
unless stromal cells are present. The stromal cells play two 
important roles: they interact directly with pro-B and pre-B 
cells, and they secrete various cytokines, notably IL-7, that 
support the developmental process. 



B-Cell Generation, Activation, and Differentiator 



249 



Immature B cells 




I3HEEP Bone-marrow stromal cells are required for matura- stromal cell, which triggers a signal, mediated by the tyrosine kinase 

tion of progenitor B cells into precursor B cells. Pro-B cells bind to activity of c-Kit, that stimulates the pro-B eel to express receptors for 

stromal cells by means of an interaction between VCAM-1 on the IL-7. IL-7 released from the stromal cell then binds to the IL-7 recep- 

stromal cell and VLA-4 on the pro-B cell. This interaction promotes tors, inducing the pro-B cell to mature into a pre-B cell. Proliferation 

the binding of c-Kit on the pro-B cell to stem cell factor (SCF) on the and differentiation evenutally produces immature B cells. 



Attheearliest developmental stage, pro-B cellsrequiredi- 
rect contact with stromal cells in the bone marrow. This in- 
teraction is mediated by several cell-adhesion molecules, 
including VLA-4 on the pro-B cell and its ligand, VCAM-1, 
on the stromal cell (Figure 11-2). After initial contact is 
made, a receptor on the pro-B cell called c-Kit interacts with 
a stromal-cell surface molecule known as stem-cell factor 
(SCF). This interaction activates c-Kit, which is a tyrosine 
kinase, and the pro-B cell begins to divide and differentiate 
into a pre-B cell and begins expressing a receptor for IL-7. 
The I L-7 secreted by the stromal cells drives the maturation 
process, eventually inducing down-regulation of the adhe- 
sion molecules on the pre-B cells, so that the proliferating 
cells can detach from the stromal cells. At this stage, pre-B 
cells no longer require direct contact with stromal cells but 
continueto require I L-7 for growth and maturation. 

Ig-Gene Rearrangment Produces 
Immature B Cells 

B-cell maturation depends on rearrangement of the immuno- 
globulin DNA in the lymphoid stem cells. The mechanisms 
of Ig-gene rearrangement were described in Chapter 5. First 
to occur in the pro-B cell stage is a heavy-chain D H -to-J H 
gene rearrangement; this is followed by a V H -to-D H jH 
rearrangement (Figure 11-3). If the first heavy-chain re- 
arrangement is not productive, then V H -D H -J H rearrange- 



ment continues on the other chromosome. Upon completion 
of heavy-chain rearrangement, the eel I isclassified asa pre-B 
cell. Continued development of a pre-B cell into an imma- 
ture B cell requires a productive light-chain gene rearrange- 
ment. Becauseof allelic exclusion, only onelight-chain isotype 
is expressed on the membrane of a B cell. Completion of a 
productive light-chain rearrangement commits the now im- 
mature B cell to a particular antigenic specificity determined 
by the cell's heavy-chain VDJ sequence and light-chain VJ 
sequence. ImmatureB cells express ml gM (membranelgM) 
on the eel I surface. 

As would be expected, the recombinase enzymes RAG-1 
and RAG-2, which are required for both heavy-chain and 
light-chain gene rearrangements, are expressed during the 
pro-B and pre-B cell stages (see Figure 11-3). The enzyme 
terminal deoxyribonucleotidyl transferase (TdT), which cat- 
alyzes insertion of N-nucleotidesattheD H -jH and V H -D H - 
J H coding joints, is active during the pro-B cell stage and 
ceases to be active early in thepre-B-cell stage. BecauseTdT 
expression is turned off during the part of the pre-B-cell 
stage when light-chain rearrangement occurs, N-nucleotides 
are not usually found in theV L -J L coding joints. 

The bone-marrow phase of B-cell development culmi- 
natesintheproductionof anlgM-bearingimmatureBcell.At 
thisstageof development the B cell isnot fully functional, and 
antigen induces death or unresponsiveness (anergy) rather 
than division and differentiation. Full maturation is signaled 



250 



Generation of B-Cell and T-Cell Responses 



Bone marrow 
antigen-independent 

Surrogate light 

chain of pre-BCR IgM 




NAIVE B CELL MATURE B CELL 



H-chain genes Germ line 
L-chain genes Germ line 



RAG-1/ 2 
TdT 



DhJh 

Surrogate 
Vp re-Band X5 
Germ-line 
KandX 



Surrogate 
light chain 



V H D H J H 

Surrogate 
Vpre-B and X5 



Surrogate 

light chain 



CD45R, CD19, 
HSA(CD24), — 
lg-a/lg-(5 



mlgM - 



mlgD 



■J[ctun**y Sequence of events and characteristics of the stages thesis of both membrane-bound IgM and IgD by mature B cells, 

in B-cell maturation in the bone marrow. The pre-B cell expresses a RAG-1/2 =two enzymes encoded by recombination-activating genes; 

membrane immunoglobulin consisting of a heavy (H ) chain and sur- TdT =terminal deoxyn bo nucleotidyl transferase. A number of B-cell- 

rogate light chains, Vpre-B and \5. Changes in the RN A processing associated transcription factors are important at various stages of 

of heavy-chain transcripts following the pre-B cell stage lead to syn- B-cell development; some are indicated here. 



by the co-expression of IgD and IgM on the membrane. This 
progression involves a change in RNA processing of the 
heavy-chain primary transcript to permit production of two 
mRNAs, one encoding the membrane form of the u, chain 
and the other encoding the membrane form of the 8 chain 
(seeFigure 5- 19). Although IgD isa characteristic cell-surface 
marker of mature naive B cells, its function is not clear. How- 
ever, since immunoglobulin 8 knockout micehave essentially 
normal numbers of fully functional B cells, IgD is not essen- 
tial to either B-cell development or antigen responsiveness. 



The Pre-B-Cell Receptor Is Essential 
for B-Cell Development 

As we saw in Chapter 10, during one stage in T-cell develop- 
ment, the B chain of theT-cell receptor associates with pre- 
Ta to form the pre-T-cell receptor (see Figure 10-1). A 
parallel situation occurs during B-cell development. In the 
pre-B cell, the membrane |x chain is associated with the sur- 
rogate light chain, a complex consisting of two proteins: a 
V-like sequence called Vpre-B and a C-like sequence called 



B-Cell Generation, Activation, and Differentiator 



Immature B cell 
VhDhJhC^ 




StopsV H ^D H JH 
(allelic exclusion) ? 



Induce 



~*J K - 



m«mjj]|P • Schematic diagram of sequential expression of mem- 
brane immunoglobulin and surrogate light chain at different stages 
of B-cell differentiation in the bone marrow. The pre-B-cell receptor 
contains a surrogate light chain consisting of a Vpre-B polypeptide 



and a \5 polypeptide, which are noncovalently associated. The im- 
mature B cell no longer expresses the surrogate light chain and in- 
stead expresses the k or \ light chain together with the jjl heavy 
chain. 



\5, which associate noncovalently to form alight-chain-like 
structure. 

The membrane- bound complex of (jl heavy chain and sur- 
rogate light chain appearson thepre-B cell associated with the 
Ig-a/lg-B heterodimertoformthepre-B-cell receptor (Figure 
11-4). Only pre-B cells that are able to express membrane- 
bound |jl heavy chains in association with surrogate light 
chainsareableto proceed along the maturation pathway. 

There is speculation that the pre-B-cell receptor recog- 
nizes a not-yet-identified ligand on the stromal-cell mem- 
brane, thereby transmitting a signal to the pre-B cell that 
preventsV H toD H jH rearrangement of theother heavy-chain 
allele, thus leading to allelic exclusion. Following the estab- 
lishment of an effective pre-B-cell receptor, each pre-B cell 
undergoes multiplecell divisions, producing 32 to 64 descen- 
dants. Each of these progeny pre-B cells may then rearrange 
different light-chain gene segments, thereby increasing the 
overall diversity of the antibody repertoire. 

The critical role of the pre-B-cell receptor was demon- 
strated with knockout micein which thegeneencodingthe\5 
protein of the receptor was disrupted. B-cell development in 
these mice was shown to be blocked at the pre-B stage, which 
suggests that a signal generated through the receptor is neces- 
sary for pre-B eel Is to proceed to the immature B-cell stage. 

Knockout Experiments Identified Essential 
Transcription Factors 

As described in Chapter 2, many different transcription fac- 
tors act in the development of hematopoietic cells. Nearly a 
dozen of them have so far been shown to play roles in B-cell 
development. Experiments in which particular transcription 



factors are knocked out by gene disruption have shown that 
four such factors, E2A, early B-cell factor (EBF), B-ceJI- 
specific activator protein (BSAP), and Sox-4are particularly 
important for B-cell development (see Figure 11-3). Mice 
that lack E2A do not express RAG-1, are unable to make 
D H J H rearrangements, and fail to express \5, a critical com- 
ponent of thesurrogate light chain. A similar pattern isseen 
in EBF-deficient mice. These findings point to important 
roles for both of these transcription factors early in B-cell 
development, and they may play essential roles in the early 
stagesof commitmenttotheB-cell lineage. Knockingout the 
Pax-5 gene, whose product is the transcription factor BSAP, 
also results in the arrest of B-cell development at an early 
stage. Binding sites for BSAP are found in the promoter re- 
gions of a number of B-cell-specific genes, including Vpre-B 
andx.5, in a number of Ig switch regions, and in thelg heavy- 
chain enhancer. This indicates that BSAP plays a role beyond 
the early stages of B-cell development. This factor is also ex- 
pressed in the central nervous system, and its absence results 
in severe defects in mid-brain development. Although theex- 
act site of action of Sox-4 is not known, it affects early stages 
of B-cell activation. While Figure 11-3 shows that all of these 
transcription factors affect development at an early stage, 
so m e of th em are acti ve at I ater stages al so . 

Cell-Surface M arkers Identify 
Development Stages 

The developmental progression from progenitor to mature 
B cell istypified by a changing pattern of surf ace markers (see 
Figure 11-3). At the pro-B stage, the cells do not display the 
heavy or light chainsof an ti body but they do expressCD45R, 



252 



Generation of B-Cell and T-Cell Responses 



which is a form of the protein tyrosine phophatase found on 
leukocytes, and the signal-transducing molecules lg-a/lg-p, 
which are found in association with the membrane forms of 
antibody in later stages of B-cell development. Pro-B cells 
also express CD 19 (part of the B-cell coreceptor), CD43 
(leukosialin), and CD24, a molecule also known as heat- 
stable antigen (H SA) on the surface. At this stage, c-Kit, a re- 
ceptor for a growth- promoting ligand present on stromal cells, 
is also found on the surface of pro-B cells. As cells progress 
from the pro-B to the pre-B stage, they express many of the 
samemarkersthatwerepresentduringthepro-B stage; how- 
ever, they cease to express c-Kit and CD43 and begin to ex- 
press CD25, the a chain of the I L- 2 receptor. The display of 
the pre-B-cell receptor (pre-BCR) is a salient feature of the 
pre-B cell stage. After rearrangement of the light chain, sur- 
faceimmunoglobulin containing both heavy and light chains 
appears, and thecells, now classified asimmatureB cells, lose 
thepre-BCR and no longer express CD 25. Monoclonal anti- 
bodies are available that can recognize all of these antigenic 
markers, making it possibleto recognize and isolate thevari- 
ous stages of B-cell development by the techniques of im- 
munohistology and flow cytometry described in Chapter 6. 

B-l B Cells Are a Self-Renewing B-Cell Subset 

There is a subset of B cells, called B-l B cells, that arise before 
B-2 B cells, the major group of B cells in humansand mice. In 
humans and mice, B-l B cells compose about 5% of the total 
B-cell population. They appear during fetal life, express sur- 
face I gM but little or no I gD, and are marked by the display of 
CD5. However, CD5isnot an indisp en sablecomponent of the 
B-l lineage, it doesnot appear on theB-lcdlsof rats, and mice 
that lack afunctional CD5 genestill produce B-l cells. In ani- 
mals whose B-2 B cells are the major B-cell population, B-l 
cellsareminor populationsin such secondary tissues as lymph 
nodes and spleen. Despite their scarcity in many lymphoid 
sites, they are the major B-cell typefound in the peritoneum. 
Although there is not a great deal of definitive informa- 
tion on the function of B-l cells, several features set them 
apart from the B-2 B cells of humansand mice. Duringfetal 
life, B-l cells arise from stem cells in bone marrow. However, 
in postnatal life this population renews itself by the prolifer- 



ation of some B-l cells in sites outside the bone marrow to 
form additional naiveB-lcells.TheB-1 population responds 
poorly to protein antigens but much better to carbohydrate 
ones. Most of its members are I gM -bearing cells, and this 
population undergoesmuch less somatic hypermutation and 
classswitchingthantheB-2setof Bcellsdoes. Consequently, 
theantibodiesproduced by a high proportion of B-l eel I s are 
of rather low affinity. 

Self-Reactive B Cells Are Selected Against 
in Bone Marrow 

It is estimated that in the mouse the bone marrow produces 
about 5 x 10 7 B cells/day but only 5 x 10 6 (or about 10%) 
are actually recruited into the recirculating B-cell pool. This 
meansthat90% of the B cells produced each daydiewithout 
ever leaving the bone marrow. Some of this loss is attribut- 
able to negativeselection and subsequent elimination (clonal 
deletion) of immature B cells that express auto-antibodies 
against self-antigens in the bone marrow. 

It haslong been established that thecrosslinkageof mlgM 
on immature B cells, demonstrated experimentally by treat- 
ing immature B cells with antibody against the inconstant re 
gion, can cause the cells to die by apoptosis within the bone 
marrow. A similar process is thought to occur in vivo when 
immatureBcellsthatexpressself-reactivemlgM bind to self- 
antigens in the bone marrow. For example, D. A. Nemazee 
and K.Burki introduced atransgeneencodingtheheavy and 
light chainsof an IgM antibody specific for K k , an H-2 fc class 
I MHC molecule, into H-2 d and H-2 dA mice(Figurell-5a,b). 
Sincedassl MHC molecules are expressed on themembrane 
of all nucleated cells, the endogenous H-2 fc and H-2 d classl 
MHC molecules would be present on bonemarrow stromal 
cells in the transgenic mice. In the H-2 d mice, which do not 
express K k , 25%-50% of the mature, peripheral B cells ex- 
pressed thetransgeneencoded anti-K^ both as a membrane 
antibody and as secreted antibody. In contrast, in theH-2 dA 
mice, which express K k , no mature, peripheral B cells ex- 
pressed thetransgeneencoded antibody to H-2 k (Table 11-1). 
These results suggest that there is negative selection against 
any immature B cells expressing auto-antibodies on their 
membranes because these antibodies react with self-antigen 



EXPRESSION OFTRANSGENE 



Experimental animal 

Nontransgenics 
H-2 01 transgenics 
H^ ^ transgenics 

SOURCE: Adapted from D. A 



N umber of animals tested 



AsmembraneAb 



(+) 
(-) 



<0.3 

93.0 



B-Cell Generation, Activation, and Differentiator 



, 11 253 



(a) H-2dlk transgenics 



Immature B cells 




| Experimental evidence for negative selection (clonal 
deletion) of self-reactive B cells during maturation in the bone mar- 
row. The presence or absence of mature peripheral B cells expressing 
a transgene-encoded IgM against the H-2 class I molecule K^was 
determined in H -2 d/k mice (a) and H-2 d mice (b). In the U-2 d/k trans- 
genics, the immature B cells recognized the self-antigen K^and were 
deleted by negative selection. In the H-2 d transgenics, the immature 
B cells did not bind to a self-antigen and consequently went on to 



A few mature B cells with new 
light chains no longer bind K k 

mature, so that 25%-50% of the splenic B cells expressed the trans- 
gene-encoded anti-K*as membrane Ig. M ore detailed analysis of the 
H -2 d/k transgenics revealed a few peripheral B cells that expressed the 
transgene-encoded |x chain but a different light chain (c). Apparently 
a few immature B cells underwent light-chain editing, so they no 
longer bound the K k molecule and consequently escaped negative se- 
lection. [Adapted from D.A. NemazeeandK. Burki, 1989, N ature337: 
562;S. L Tiegsetal., 1993, JEM 177:1009.] 



(eg., the K^ molecule in H -2 d/k transgenics) present on stro- 
mal cells, leading to crosslinking of the antibodies and subse- 
quent death of the immature B cells. 

Self-Reactive B Cells M ay Be Rescued 
by Editing of Light-Chain Genes 

Later work usingthetransgenicsystem described byNemazee 
and Burki showed that negative selection of immature B cells 



does not always result in their immediate deletion (Figure 
11- 5c). Instead, maturation of the self- reactive eel I isarrested 
while the B cell "edits" the light-chain gene of its receptor. 
In this case, the H-2** transgenics produced a few mature 
B eel Is that expressed mlgM containing the |x chain encoded 
in thetransgene, but different, endogenous light chains. One 
explanation for these results is that when some immature 
B cells bind a self-antigen, maturation is arrested; the cells 
up-regulate RAG- land RAG-2 expression and begin further 



254 



Generation of B-Cell and T-Cell Responses 



rearrangement of their endogenous light-chain DNA. Some 
of these eel Is succeed in replacing theK light chain of the self- 
antigen reactive antibody with a X chain encoded by endoge- 
nous\-chain gene segments. As a result, these eel Is will begin 
to express an "edited" m I gM with a different light chain and a 
specificity that is not self-reactive. These cells escape negative 
selection and leave the bone marrow. 



B-Cell Activation and Proliferation 

After export of B cellsfrom thebonemarrow, activation, pro- 
liferation, and differentiation occur in the periphery and re- 
quireanti gen. Antigen-driven activation and clonal selection 
of naiveB cells leadsto generation of plasma cells and mem- 
ory B cells. In the absence of antigen-induced activation, 
naive B cells in the periphery have a short life span, dying 
within a few weeks by apoptosis (see Figure 11-1) . 

Thymus-Dependent and Thymus- 
Independent Antigen H ave Different 
Requirements for Response 

Depending on the nature of the antigen, B-cell activation pro- 
ceeds by two different routes, onedependent upon T H cells, the 
other not. The B-cell response to thymus-dependent (TD ) an- 
tigens requires direct contact with T H cells, not simply expo- 
sure to T H -derived cytokines. Antigens that can activate B cells 
in theabsenceof thiskind of direct participation byT H cellsare 
known asthymus-independent(TI)antigens.TI antigensare 
divided into types 1 and 2, and they activate B cells by different 
mechanisms. Some bacterial cell-wall components, including 
lipopolysaccharide(LPS), function as typel thymus-independent 
(Tl-l) antigens. Type2 thymus-independent (TI-2) antigensare 
highly repetitious molecules such as polymeric proteins (eg., 
bacterial flagellin) or bacterial cell-wall polysaccharides with 
repeating polysaccharide units. 

M ost Tl - 1 antigens are polyclonal B-cell activators (mito- 
gens); that is, they are able to activate B cells regardless of 
their antigenic specificity. At high concentrations, someTI-1 
antigens will stimulate proliferation and antibody secretion 
by as many as one third of all B cells. The mechanism by 
which Tl-l antigens activate B cells is not well understood. 
When B cells are exposed to lower concentrations of Tl-l 
anti gens, only thoseB cellsspecific for epitopesoftheanti gen 
will be activated. These antigenscan stimulate antibody pro- 
duction in nude mice (which lack a thymus and thus are 
greatly deficient in T cells), and the response is not greatly 
augmented by transferring T cells into these athymic mice, 
indicating that Tl-l antigens are truly T-cell independent. 
The prototypic Tl-l antigen islipopolysaccharide(LPS), a 
major component of the cell wallsof gram-negative bacteria. 
At low concentrations, LPS stimulates the production of 
antibodies specific for LPS. At high concentrations, it is a 
polyclonal B-cell activator. 



TI-2 antigens activate B cells by extensively crosslinking 
the mlg receptor. However, TI-2 antigens differ from Tl-l 
antigens in three important respects. First, they are not B-cell 
mitogens and so do not act as polyclonal activators. Second, 
Tl-lantigenswill activate both matureandimmatureB cells, 
but TI-2 antigens activate mature B cells and inactivate im- 
mature B cells. Third, although the B-cell response to TI-2 
antigens does not require direct involvement of T H cells, 
cytokines derived from T H cells are required for efficient 
B-cell proliferation and for class switching to isotypes other 
than IgM .Thiscan be shown bycomparingtheeffectof TI-2 
antigens in mice made T-cell- deficient in various ways. In 
nude mice, which lack thymus-derivedT cells but do contain 
a few Tcellsthat arise from other sites that probably liein the 
intestine, TI-2 antigens do elicit B-cell responses. TI-2 anti- 
gens do not induce antibody production in mice that cannot 
express eitherap or 78 TCRs because thegenes en coding the 
TCR (3 and 8 chains have been knocked out. Administration 
of a few T cells to these TCR-knockout mice restores their 
ability to elicit B-cell responses toTI-2 antigens. 

Thehumoral response to thymus-independent anti gens is 
different from the response to thymus-dependent antigens 
(Table 11-2). Theresponse to Tl antigens is generally weaker, 
no memory cells are formed, and IgM is the predominant 
antibody secreted, reflecting a low level of class switching. 
These differences highlight the important role played by T H 
cells in generating memory B cells, affinity maturation, and 
class switching to other isotypes. 

Two Types of Signals Drive B Cells into 
and Through the Cell Cycle 

Naive, or resting, BcellsarenondividingcellsintheGoStageof 
the cell cycle. Activation drives the resting cell into the cell cy- 
cle, progressing through Gi into theS phase, in which DNA is 
replicated. The transition from Gi to Sis a critical restriction 
point in the cell cycle. Once a cell has reached S, it completes 
thecell cycle, movingthrough G 2 and into mitosis(M ). 

Analysisof the progression of lymphocytes from G to the 
S phase revealed similarities with the parallel sequence in fi- 
broblast cells. These events could be grouped into two cate- 
gories, competence signals and progression signals. Compe- 
tence signalsdrive the B cell from G into early Gi, rendering 
the cell competent to receive the next level of signals. Pro- 
gression signals then drive the cell from Gi into S and ulti- 
mately to cell division and differentiation. Competence is 
achieved by not one but two distinct signaling events, which 
are designated signal 1 and signal 2. These signaling events are 
generated by different pathways with thymus-independent 
and thymus-dependent antigens, but both pathways include 
signals generated when multivalent antigen bindsand cross- 
links mlg (Figure 11-6). OncetheB cell has acquired an ef- 
fective competence signal in early activation, the interaction 
of cytokines and possibly other ligandswith theB-cell mem- 
brane receptors provides progression signals. 



B-Cell Generation, Activation, and Differentiator 



Chemical nature 

Humoral response 
Isotype switching 
Affinity maturation 
Immunologic memory 
Polyclonal activation 



Tl ANTIGENS 
Type 2 



Bacterial cell-wall 
components (e.g., LPS) 



Polymeric protein antigens; 
capsular polysaccharides 



Transduction of Activating Signals Involves 
I g-a/ 1 g-p H etero d i m ers 

For many years, immunologistsquestioned how engagement 
of the I g receptor by antigen could activate intracellular sig- 
naling pathways. All isotypes of mlg have very short cyto- 
plasmic tails. Both ml gM andmlgD on Bcellsextend into the 
cytoplasm by only three amino acids; the mlgA tail consists 
of 14 amino acids; and the ml gG and ml gE tails contains 28 
amino acids. In each case, the cytoplasmic tail istoo short to 
be able to generate a signal by associating with intracellular 
signaling molecules, such as tyrosine kinases and G proteins. 
Thediscoverythat membrane I g is associated with thedisul- 
fide-linked heterodimer Ig-a/lg-B, forming theB-cell recep- 
tor(BCR), solved this longstanding puzzle. Though it was 
originally thought that two Ig-a/lg-B heterodimers associ- 
ated with one mlg to form the B-cell receptor, careful bio- 
chemical analysis has shown that only one Ig-a/lg-B net- 



fa) Tl-l antigen (b) TD antigen 




An effective signal for B-cell activation involves two 
distinct signals induced by membrane events. Binding of a type 1 
thymus-independent (Tl-l) antigen to a B cell provides both signals. 
A thym us-dependent (TD) antigen provides signal 1 bycrosslinking 
mlg, but a separate interaction between CD40 on the B cell and 
CD40Lon an activated T H cell is required to generate signal 2. 



erodimer associates with a single mlg molecule to form the 
receptor complex. (Figure 11-7). ThustheBCR isfunction- 
ally divided into the ligand-binding immunoglobulin mole- 
cule and the signal-transducing Ig-a/lg-B heterodimer. A 
similar functional division marks the pre-BCR, which trans- 
duces signals via a complex consisting of an Ig-a/lg-B het- 
rodimer and |jl heavy chains combined with the surrogate 
light chain (seeFigure 11-4). Thelg-a chain hasalongcyto- 
plasmic tail containing 61 amino acids; the tail of thelg-B 
chain contains 48 amino acids. The cytoplasmic tails of both 
lg-a and Ig-B contain the 18-residue motif termed the 
immunoreceptor tyrosine-based activation motif (ITAM; 
see Figure 11-7) which has already been described in several 
moleculesoftheT-cell-receptor complex (see Figure 10-11). 
Interactions with the cytoplasmic tails of Ig-a/lg-p trans- 
duce thestimulus produced bycrosslinking of mlg molecules 
into an effective intracellular signal. 

I n the BCR and theTCR, as well as in a number of recep- 
tors for the Fc regions of particular I g classes (FceRI for IgE; 
FC7RIIA, FC7RIIC, FC7RIIIA for IgG), ligand binding and 
signal transduction are mediated by a multimeric complex of 
proteinsthat isfunctionally compartmentalized. The ligand- 
binding portions of these complexes (mlg in the case of the 
BCR) is on the surface of the cell, and the signal-transducing 
portion is mostly or wholly within the cell. As is true of the 
TCR,signalingfromtheBCRismediated by protein tyrosine 
kinases (PTKs). Furthermore, like theTCR, the BCR itself 
has no PTK activity; this activity is acquired by recruitment 
of a number of different kinases, from nearby locations within 
thecell, to thecytopiasmictailsof thesignal. Phosphorylation 
of tyrosines within the ITAM sof the BCR by receptor associ- 
ated PTKs is among the earliest events in B-cell activation 
and plays a key role in bringing other critical PTKs to the 
BCR and in their activation. The antigen-mediated crosslink- 
ing of BCRs initiates a number of signaling cascades that ulti- 
mately result in the cell's responses to the crosslinking of its 
surfaceimmunoglobulinbyantigen.ThecrosslinkingofBCRs 
resultsin theinduction of many si gnal-tr an sduction pathways 



256 



Generation of B-Cell and T-Cell Responses 



Crosslinked 
Bcell 




I The initial stages of signal transduction by an activated 
B-cell receptor (BCR). The BCR comprises an antigen-binding mlg 
and one signal-transducing Ig-a/lg-p heterodimer Following antigen 
crosslinkage of the BCR, the immunoreceptortyrosine-based activation 
motifs (ITAMs) interact with several members oftheSrc family of tyro- 
sine kinases (Fyn, Blk, and Lck), activating the kinases. The activated 



enzymes phosphoryiate tyrosine residues on the cytoplasmic tails of 
the Ig-a/lg-p heterodimer creating docking sites forSyk kinase, which 
is then also activated. The highlyconserved sequence motif of ITAM s is 
shown with the tyrosines (Y) in blue. D/E indicates that an aspartate or 
a glutamate can appear at the indicated position, and X indicates that 
the position can be occupied by any amino acid. 



and theactivati on of theB cell. Figure ll-8showsmany paral- 
lels between B-cell andT-cell activation. Theseinclude: 

s Compartmentalization of function within receptor 
subunits. Both the B-cell and T-cell pathways begin with 
antigen receptors that are composed of an antigen- 
binding and a signaling unit. The antigen-binding unit 
confers specificity, but has cytoplasmic tailstoo short to 
transduce signals to the cytoplasm of thecell. The 
signaling unit has long cytoplasmic tails that are the 
signal transducers of the receptor complex. 

Activation by membrane- associated Src protein tyrosine 
kinases The receptor-associated PTKs(Lck in T cells and 
Lyn, Blk, and Fyn in B cells) catalyze phosphorylations 
during the early stages of signal transduction that are 
essential to theformation of a functional receptor 
signaling complex. 

s Assembly of a large signaling complex with protein- 
tyrosine-kinase activity: The phosphorylated tyrosines in 
the ITAMs of the BCR andTCR providedockingsitesfor 
the molecules that endow these receptors with PTK 
activity; ZAP- 70 in T cells and Syk in B cells. 

s Recruitment of other signal-transduction pathways 
Signalsfrom the BCR andTCR result in theproduction 



of the second messengers I P 3 and DAG. I P 3 causes the 
release of Ca 2+ from intracellular stores, and DAG 
activates PKC. A third important set of signaling 
pathways are those governed by thesmall G proteinsRas 
and Rac that are also activated by signals received 
through theTCR or BCR. 

• Changesin geneexpression: Oneof theimportant 
outcomes of signal-transduction processes set in motion 
with engagement of the BCR or theTCR isthe 
generation or translocation to the nucleus of active 
transcription factors that stimulate or inhibitthe 
transcription of specific genes. 

Failures in signal transduction can have severe conse- 
quences for the immune system. The Clinical Focus on 
X-linked agammaglobulinemia describes the effect of defec- 
tive signal transduction on the development of B cells. 

The B-Cell-Coreceptor Complex Can 
Enhance B-Cell Responses 

Stimulation through antigen receptors can be modified sig- 
nificantly by signals through coreceptors. Recall that co- 
stimulation through CD28 is an essential feature of effective 
positive stimulation of T lymphocytes, while signaling 
through CTLA-4 inhibits theresponseoftheT cell. In B cells 



B-Cell Generation, Activation, and Differentiator 



VI SUALIZI N G CONCEPTS 




Small G-protein mediated mediated 

pathways pathways pathways 



• Changes in pattern 


of gene expression 


• Functional changes 


in cells 


• Differentiation 




• Activation 





Some of the many signal-transduction pathways 
activated by the BCR. In one pathway Syk activates PLC72 by tyro- 
sine phosphorylation. PLC72 then hydrolyzes PIP 2 , a membrane 
phospholipid, to produce the second messengers DAG and IP 3 . 
DAG and Ca 2+ released bythe action of IP 3 collaboratively activate 



the PKC, which induces additional signal-transduction pathways. 
The activated receptorcomplexalso generates signals that activate 
the Ras pathway Activated Ras initiates a cascade of phosphoryla- 
tions that culminates in the activation of transcription factors that 
up-regulate the expression of many genes. 



5 mat 



a component of the B-cell membrane, called the B-cell core- 
ceptor, provides stimulatory modifying signals. 

TheB-cellcoreceptorisa complex of three proteins: CD 19, 
CR2 (CD21), and TAPA-1 (CD81) (Figure 11-9). CD19, a 
member of the immunoglobulin superfamily, hasa long cyto- 
plasmic tail and three extracellular domains. TheCR2 compo- 
nent is a receptor of C3d, a breakdown product of the 
complement system, which is an important effector mecha- 
nism for destroying invaders (Chapter 13); note that the in- 
volvement of C3d in the pathway for coreceptor activity 
reveals different arms of the immune system interacting with 
each other. CR2 also functions as a receptor for a membrane 
moleculeand thetransmembraneprotein TAPA-1. 1 n addition 
to the stimulatory coreceptor, another molecule, CD22, which 



isconstitutively associated with theB-cell receptor in resting B 
cells, deliversa negative signal that makes B-cellsmoredifficult 
to activate. As shown in Figure 11-9, the CR2 component of 
the coreceptor complex binds to complement-coated antigen 
that has been captured bythemlgontheB cell. Thiscrosslinks 
thecoreceptor to theBCR and allows theCD 19 component of 
thecoreceptor to interactwith thelg-a/lg-p component of the 
BCR. CD 19 contains six tyrosine residues in its long cytoplas- 
mictail and isamajor substrate of the protein tyrosine kinase 
activity that is mediated by crosslinkageof theBCR. Phospho- 
rylation of CD19 permits it to bind a number of signaling 
molecules, including the protein tyrosine kinase Lyn. 

Thedelivery of these si gnalingmoleculesto theBCR com- 
plex contributes to theactivation process, and thecoreceptor 



258 



Generation of B-Cell and T-Cell Responses 



CLI N I CAL FOCUS 



X-Linked Agammaglobulinemia: 
A Failure in Signal Transduction 
and B-Cell Development 



X-linked 



agammaglobu- 
linemia is a genetically determined im- 
munodeficiency disease characterized by 
the inability to synthesize all classes of 
antibody It was discovered in 1952 by 
0. C. Bruton in what is still regarded as 
an outstanding example of research in 
clinical immunology Bruton's investiga- 
tion involved a young boy who had 
mumps 3 times and experienced 19 dif- 
ferent episodes of serious bacterial infec- 
tions during a period of just over 4 years. 
Because pneumococcus bacteria were 
isolated from the child's blood during 



10 of the episodes of bacterial infection, 
attempts were made to induce immunity 
to pneumococcus by immunization with 
pneumococcus vaccine. The failure of 
these efforts to induce antibody responses 
prompted Bruton to determine whether 
the patient could mount antibody re- 
sponses when challenged with other anti- 
gens. Surprisingly immunization with 
diphtheria and typhoid vaccine prepara- 
tions did not raise humoral responses in 
this patient. Electrophoretic analysis of the 
patient's serum revealed that although 
normal amounts of albumin and other typ- 
ical serum proteins were present, gamma 



globulin, the major antibody fraction of 
serum, was absent. Having traced the 
immunodeficiency to a lack of antibody 
Bruton tried a bold new treatment. H e ad- 
ministered monthly doses of human im- 
mune serum globulin. The patient's ex- 
perience of a fourteen-month period free 
of bacterial sepsis established the useful- 
ness of the immunoglobulin replacement 
for the treatment of immunodeficiency. 

Though initially called Bruton's agam- 
maglobulinemia, this hereditary immun- 
odeficiency disease was renamed X-linked 
agammaglobulinemia, orX-LA, after the 
discovery that the responsible gene lies 
on the X chromosome. The disease has 
the following clinical features: 

Because this defect is X-linked, 
almost all afflicted individuals are 
male. 

s Signs of immunodeficiency may 
appear as early as 9 months 
after birth, when the supply of 




l^cjjy^lQ Tne B ~ ce " coreceptor is a complex of three cell mem- 
brane molecules: TAPA-1 (CD81), CR2 (CD21), and CD19. Binding of 
the CR2 component to complement-derived C3d that has coated anti- 
gen captured bymlg results in the phosphorylation of CD 19. The Src- 
familytyrosine kinase Lyn binds to phosphorated CD19. The resulting 
activated Lyn and Fyn can trigger the signal-transduction pathways 
shown in Figure 11-8 that begin with phospholipase C. 



complex serves to amplify the activating signal transmitted 
through the BCR. In one experimental in vitro system, for 
example, 10 4 molecules of mlgM had to be engaged by anti- 
gen for B-cell activation to occur when the coreceptor was 
not involved. But when CD19/CD2/TAPA-1 coreceptor was 
crosslinked to the BCR, only 10 2 molecules of mlgM had to 
be en gaged for B-cell activation. Another striking experiment 
highlights the role played by theB-cell coreceptor. Mice were 
immunized with either unmodified lysozyme or a hybrid 
protein in which genetic engineering was used to join hen's 
egg lysozymeto C3d. Thefusion protein bearing 2 or 3 copies 
of C3d produced anti-lysozyme responses that were 1000 to 
10,000 times greater than those to lysozyme alone. Perhaps 
coreceptor phenomena such as these explain how naive 
B eel Is that often express ml g with low affinity for antigen are 
ableto respond to low concentrationsof antigen in a primary 
response. Such responses, even though initially of low affin- 
ity, can play a significant role in the ultimate generation of 
high-affinity antibody. As described later in thischapter, re- 
sponse to an antigen can lead to affinity maturation, result- 
ing in higher average affinity of theB-cell population. Finally, 
two experimental observations indicate that the CD 19 com- 
ponent of the B-cell coreceptor can play a role independent 
of CR2, thecomplement receptor. I n normal mice, artifically 
crosslinkingtheBCR with anti-BCRantibodiesresultsin the 



B-Cell Generation, Activation, and Differentiator 



259 



maternal antibody acquired in 
utero has decreased below 
protective levels. 

■ There is a high frequency of 
infection by Streptococcus 
pneumoniae and Haemophilus 
influenzae; bacterial pneumonia, 
sinusitis, meningitis, or septicemia 
are often seen in these patients. 

Although infection by many viruses 
is no more severe in these patients 
than in normal individuals, long- 
term antiviral immunity is usually 
not induced. 

Analysis by fluorescence microscopy 
or flow cytometry shows few or no 
mature B cells in the blood. 

Studies of this disease at the cellular 
and molecular level provide insights into 
the workings of the immune system. A 
scarcity of B cells in the periphery ex- 
plained the inability of X-LA patients to 



make antibody Studies of the cell popula- 
tions in bone marrow traced the lack of B 
cells to failures in B-cell development. The 
samples displayed a ratio of pro-B cells to 
pre-B cells 10 times normal, suggesting 
inhibition of the transition from the pro- 
to the pre-B-cell stage. The presence of 
very few mature B cells in the marrow indi- 
cated a more profound blockade in the de- 
velopment of B cells from pre-B cells. 

In the early 1990s, the gene responsi- 
ble for X-I.A was cloned. The normal coun- 
terpart of this gene encodes a protein 
tyrosine kinase that has been named Bru- 
ton's tyrosine kinase (Btk) in honor of the 
resourceful and insightful physician who 
discovered X-LA and devised a treatment 
for it. Parallel studies in mice have shown 
that the absence of Btk causes a syn- 
drome known as xid, an immunodefi- 
ciency disease that is essentially identical 
to its human counterpart, X-LA. Btk has 
turned out to play important roles in B-cell 
signaling. For example, crosslinking of the 



B-cell receptor results in the phosphoryla- 
tion of a tyrosine residue in the catalytic 
domain of Btk. This activates the protein- 
tyrosine-kinase activity of Btk, which then 
phosphoryiates phospholipaseC-72 (PLC- 
72); in vitro studies of cell cultures in 
which Btk has been knocked out show 
compromised PLC-y 2 activation. Once 
activated, PLC-y 2 hydrolyzes membrane 
phospholipids, liberating the potent sec- 
ond messengers IP 3 and DAG. As men- 
tioned earlier, IP 3 causes a rise in in- 
tracellular Ca 2+ , and DAG is an activatorof 
protein kinase C (PKC). Thus, Btk plays a 
pivotal role in activating a network of in- 
tracellular signals vital to the function of 
mature B cells and earlier members of the 
B-cell lineage. Research has shown that it 
belongs to a family of PTKs known as Tec 
kinases; its counterpart in T cells is Itk 
The insights gained from studies of X-LA, 
xid, and Btk are impressive examples of 
how the study of pathological states can 
clarify the workings of normal cells. 



stimulation of some of the signal-transduction pathways 
characteristic of B-cell activation. On the other hand, treat- 
ment of B cellsfrom micein which CD19 has been knocked 
out with anti-BCR antibody fails to induce these pathways. 
Furthermore, CD 19 knockout mice makegreatly diminished 
antibody response to most antigens. 

T H Cells Play Essential Roles in Most 
B-Cell Responses 

As noted already, activation of B cells by soluble protein anti- 
gens requiresthe involvement of T H cells. Binding of antigen 
to B-cell mlg does not itself induce an effective competence 
signal without additional interaction with membrane mole- 
cules on theT H cell. In addition, a cytokine- mediated pro- 
gression is required for B-cell proliferation. Figure 11-10 out- 
lines the probable sequence of events in B-cell activation by 
a thymus-dependent (TD) antigen. This process is consid- 
erably more complex than activation induced by thymus- 
independent (Tl) antigens. 

FORMATION OF T-B CONJUGATE 
After binding of antigen by mlg on B cells, the antigen is in- 
ternalized by receptor- mediated endocytosis and processed 
within theendocytic pathway into peptides. Antigen binding 



also initiates signaling through the BCR that induces the B 
cell to up-regulate a number of cell-membrane molecules, 
including class II MHC molecules and the co-stimulatory 
ligand B7 (see Figure 11- 10a). Increased expression of both 
of these membrane proteins enhances theability of the B cell 
to function as an antigen -presenting cell in T H -cell activa- 
tion. B-cells could be regarded as helping their helpers be- 
cause the antigenic peptides produced within the endocytic 
processing pathway associate with class II M HC molecules 
and arepresented on the B-cell membranetotheT H cell, in- 
ducing its activation. It generally takes 30- 60 mi n after i nter- 
nalization of antigen for processed antigenic peptides to be 
displayed on the B-cell membrane in association with class 1 1 
MHC molecules. 

BecauseaB cell recognizes and internalizes antigen specif- 
ically, by way of its membrane-bound Ig, a B cell is able to 
present antigen to T H cells at antigen concentrations that 
are 100 to 10,000 times lower than what is required for pre- 
sentation by macrophages or dendritic cells. When antigen 
concentrations are high, macrophages and dendritic cells 
are effective antigen -presenting cells, but, as antigen levels 
drop, B cells take over as the major presenter of antigen to 
T H cells. 

Once a T H cell recognizes a processed antigenic peptide 
displayed by a class 1 1 MHC molecule on the membrane of a 



260 



Generation of B-Cell and T-Cell Responses 



(a) Antigen crosslinks mlg, generating 
signal (T), which leads to increased 
expression of class II MHC and co- 
stimulatory B7. Antigen-antibody 
complexes are internalized by 
receptor-mediated endocytosis and 
degraded to peptides, some of which 
are bound by class II MHC and 
presented on the membrane as 
peptide-MHC complexes. 



(b) T H cell recognizes antigen-class II 
MHC on B-cell membrane. This plus 
co-stimulatory signal activates T H cell. 



(c) 1. T H cell begins to express CD40L 

2. Interaction of CD40 and CD40L 

provides signal (2). 

3. B7-CD28 interactions provide 
co-stimulation to theT H cell. 



(d) 1. B cell begins to express receptors 
for various cytokines. 
2. Binding of cytokines released from 
T H cell in a directed fashion sends 
signals that support the progression 
of the B cell to DNA synthesis and to 
differentiation. 




mjjjJ2lF ' Sequence of events in B-cell activation by a thymus-dependent antigen. The cell-cycle phase 
of the interacting Bcell is indicated on the right. 



B cell, the two cells interact to form aT-B conjugate (Figure 
11-11). Micrographs of T-B conjugates reveal that theT H 
cells in antigen-specific conjugates have reorganized the 
Golgi apparatus and the microtubular-organizing center to- 
ward thej unction with theB cell. This structural adjustment 
facilitates thereleaseof cytokinestowardtheanti gen-specific 
Bcell. 

CONTACT-DEPENDENT HELP MEDIATED BYCD40/CD40L 

INTERACTION 

Formation of aT-B conjugatenot only leads to the directional 

release of T H -cell cytokines, but also to the up-regulation of 



CD40L (CD154),aT H -cell membrane protein that then in- 
teracts with CD40on B cellsto providean essential signal for 
T-cell-dependent B-cell activation. CD40 belongs to thetu- 
mornecrosisfactor(TNF) family of eel I -surface protein sand 
soluble cytokines that regulate cell proliferation and pro- 
grammed cell death by apoptosis. CD40L belongstotheTNF 
receptor (TN FR) family. I nteraction of CD40L with CD40 on 
theB cell deliversa signal (signal 2) to theB cell that, in concert 
with thesignal generated by ml gcrosslinkage( signal 1), drives 
theB cell into G x (see Figure 11- 10c). The signals from CD40 
are transduced by a number of intracellular signaling path- 
ways, ultimately resulting in changesin geneexpression. Stud- 



B-Cell Generation, Activation, and Differentiator 



ies have shown that although CD40 does not have kinase ac- 
tivity, its crosslin king is followed by the activation of protein 
tyrosine kinases such as Lyn and Syk. Crosslinking of CD40 
also resultsin theactivation of phospholipaseC and thesubse- 
quent generation of the second messengers I P 3 and DAG, and 
theactivation of a number of transcription factors. Ligation of 
CD40 also results in its association with members of the 
TN FR-associated factor (TRAF) family. A consequenceof this 
interaction istheactivation of thetranscription factor NF-6B. 
Several lines of evidencehaveidentifiedtheCD40/CD40L 
interaction as the mediator of contact-dependent help. The 
roleof an inducibleT H -cell membrane protein in B-cell acti- 
vation was first revealed by experiments in which naive 
B cells were incubated with antigen and plasma membranes 
prepared from either activated or restingT H -cell clones. Only 
the membranes from the activated T H cells induced B-cell 
proliferation, suggesting that one or more molecules ex- 
pressed on the membrane of an activated T H cell engage 
receptors on the B cell to provide contact-dependent help. 
Furthermore, when antigen-stimulated B cells are treated 
with anti-CD40 monoclonal antibodies in the absence of T H 
cells, they become activated and proliferate. Thus, engage- 
ment of CD40, whether by anti bodies to CD40 or by CD40L, 
iscritical in providing signal 2 to the B cell. If appropriatecy- 
tokines are also added to this experimental system, then the 
proliferating B cells will differentiate into plasma cells. Con- 
versely, anti bodies to CD40L have been shown to block B-cell 
activation by blockingtheCD40/CD40L interaction. 

SIGNALS PROVIDED BYTh-CELL CYTOKINES 

Although B cells stimulated with membrane proteins from 

activated T H cells are able to proliferate, they fail to dif- 



fer entiateunless cytokines arealso present; thisfinding sug- 
gests that both a membrane-contact signal and cytokine 
signals are necessary to induce B-cell proliferation and dif- 
ferentiation. As noted already, electron micrographs of T-B 
conjugates reveal that the antigen-specific interaction be- 
tween aT H and a B cell induces a redistribution of T H -cell 
membrane proteins and cytoskeletal elements that results 
in the polarized release of cytokines toward the interacting 
B cell. 

Once activated, the Bee! I beginsto express membrane re- 
ceptors for various cytokines, such as IL-2, IL-4, IL-5, and 
others. These receptors then bind the cytokines produced 
by the interacting T H cell. The signals produced by these 
cytokine- receptor interactions support B-cell proliferation 
and can inducedifferentiation into plasma cellsand memory 
B cells, class switching, and affinity maturation. Each of these 
events is described in a later section. 

M ature Self-Reactive B Cells Can Be 
N egatively Selected in the Periphery 

Because some self-antigens do not have access to the bone 
marrow, B cells expressing mlgM specific for such antigens 
cannot be eliminated by the negative-selection processinthe 
bone marrow described earlier. To avoid autoimmune re- 
sponses from such mature self- reactive B cells, some process 
for deleting them or rendering them inactive must occur in 
peripheral lymphoid tissue. 

A transgenic system developed by C. Goodnow and his 
coworkers has helped to clarify the process of negative selec- 
tion of mature B cells in the periphery. Goodnow's experi- 
mental system included two groups of transgenic mice 




| Transmission electron micrographs of initial contact 

between a T cell and B cell (left) and of a T-B conjugate (right). Note the 



broad area of membrane contact between the cells after formation of 
the conjugate. [From V.M. Sanders et a/., 1986, J . Immunol. 137:2395.] 



262 



Generation of B-Cell and T-Cell Responses 





Double transgenic 
(carrying both H EL and anti-HEL transgenes) 



Anti-HEL transgenic 



Anti-HEL/HEL 
double transgenic 




IgM expression on membrane (arbitrary fluorescence 



mijjj<21^9 Goodnow's experimental system for demonstrating 
clonal anergyin mature peripheral B cells, (a) Production of double-trans- 
genic mice carry ng transgenes encoding H EL (hen egg-white lysozyme) 
and anti-H EL antibody (b) Flow cytometric analysis of peripheral B cells 
that bind H EL compared with membrane IgM levels. The number of B 
cells binding H EL was measured by determining how many cells bound 
fluorescently labeled H EL. Levels of membrane IgM were determined by 
incubating the cells with anti-mouse IgM antibody labeled with a fluores- 
cent label different from that used to label H EL. M easurement of the flu- 



orescence emitted from this label indicated the level of membrane IgM 
expressed bythe B cells. The nontransgenics (/eft) had many B cells that 
expressed high levels of surface IgM but almost no B cells that bound 
H EL above the background level of 1. Both anti-H EL transgenics (middle) 
and anti-H EL/H EL double transgenics {right) had large numbers of B 
cells that bound H EL (blue), although the level of membrane IgM was 
about twentyfold lower in the double transgenics. The data in Table 11-3 
indicate that the B cells expressing anti-H EL in the double transgenics 
cannot mount a humoral response to H EL. 



(Figure 11- 12a). One group carried a hen's egg-white lyso- 
zyme(HEL)transgenelinkedtoametallothioninepromoter, 
which placed transcription of the H EL gene under the con- 
trol of zinc levels. The other group of transgenic mice carried 
rearranged immunoglobulin heavy- and light-chain trans- 
genes encoding anti-H EL antibody; in normal mice, the fre- 
quency of H EL-reactiveB cellsison theorder of 1 in 10 3 , but 
in these transgenic mice the rearranged anti-H EL transgene 
is expressed by 60%-90% of the mature peripheral B cells. 
Goodnow mated the two groups of transgenics to produce 
"double-transgenic" offspring carrying both the HEL and 
anti-H EL transgenes. Goodnow then asked what effect H EL, 
which is expressed in the periphery but not in the bone mar- 
row, would have upon the development of B cells expressing 
theanti-H EL transgene. 

The Goodnow double-transgenic system has yielded sev- 
eral interesting findings concerning negative selection of 



B cells (Table 11-3). He found that double-transgenic mice 
expressing high levels of HEL (10~ 9 M) continued to have 
mature, peripheral B cellsbearing anti-H EL membraneanti- 
body, but these B cells were functionally nonresponsive; that 
is, they were anergic. Theflow-cytometric analysis of B cells 
from double-transgenic mice showed that, while large num- 
bers of anergic anti-H EL cells were present, they expressed 
IgM at levelsabout 20-fold lower than anti-H EL singletrans- 
genics (Figure 11- 12b). Further study demonstrated that the 
doubletransgenicshad both surface IgM and I gD, indicating 
thattheanergy wasinduced in mature rather than immature 
B cells. When these mice were given an immunizing dose of 
HEL, few anti-H EL plasma cells were induced and theserum 
anti-H EL titer was low. 

To study what would happen if a class I MHC self-antigen 
were expressed only in the periphery, Nemazee and Burki 
modified the transgenic system used in the experiments on 



B-Cell Generation, Activation, and Differentiator 



263 




Anti-H EL single transgenics 

Anti-H EL/H EL single transgenics (Group 1) 



None 
1CT 9 M 



High 
Low 



* Experimental animals were immunized with hen egg-white lysozyme (H EL). Several days later, hemolytic plaque assays for the numbei 
plasma cells secreting anti-H EL antibody were performed and the serum anti-H EL titers were determined. PFC = plaque-forming cells; 
see Figure 23-1 for a description of the plaque assay. 

SOURCE: Adapted from C. C. Goodnow, 1992, Annu. Rev. Immunol. 10:489. 



Mice with 
liver-specific 
K* transgene 



Mice with 

anti-K* 
transgene 



Double transgenics 

with K h and 
anti-K%ansgene 



Single transgenic 
(Anti-K 6 ) 



Double transgenic 
(Anti-K*/K*) 





O 


|i 




O 


o 




o 





mlgM y 

Lymph node 



mlgM > 

Lymph node 







O 


i? 






V 


o 




O 





negative selection in the bone marrow described previously 
(Figure 11- 5a). They first produced a transgene consi sting of 
the class I K b gene linked to a liver-specific promoter, so that 
the class I K b molecule could be expressed only in the liver. 
Transgenic mice expressing an anti-K b antibody on their B 
cells also were produced, and the two groups of transgenic 
mice were then mated (Figure 11- 13a). In the resulting 
double-transgenic mice, the immature B cells expressing 
anti-K b mlgM would not encounter class I K b molecules in 
the bone marrow. Flow-cytometric analysis of the B cells 
in the double transgenics showed that immature B cells 
expressing the transgene-encoded anti-K b cells were present 
in the bone marrow but not in the peripheral lymphoid 
organs (Figure 11- 13b). In the previous experiments of 
NemazeeandBurki,theclassl MHC self-antigen (H-2 fc ) was 
expressed on all nucleated cells, and immature B cells 
expressing the transgene-encoded antibody to this class I 
molecule were selected against and deleted in the bone mar- 
row (see Figure 11- 5a). In their second system, however, the 
class I self-antigen (K b ) was expressed only in the liver, so 
that negative selection and deletion occurred at the mature 
B-cell stage in the periphery. 



Tel Experimental demonstration of clonal deletion of 
self-reactive mature peripheral B cells by Nemazee and Burki. (a) Pro- 
duction of double-transgenic mice expressing the class I K b molecule 
and anti-K b antibody Because the K b transgene contained a liver- 
specific promoter, K b was not expressed in the bone marrow of the 
transgenics, (b) F I ow-cyto metric analysis of bone marrow and periph- 
eral (lymph node) B cells for K b binding versus membrane IgM 
(mlgM). In the double transgenics, B cells expressing anti-K b (blue) 
were present in the bone marrow but were absent in the lymph nodes, 
indicating that mature self-reactive B cells were deleted in the periphery 



264 



Generation of B-Cell and T-Cell Responses 



The H umoral Response 

This section considers the differences between the primary 
and secondary humoral response and the use of hapten- 
carrier conjugates in studying the humoral response. 

Primary and Secondary Responses Differ 
Significantly 

The kinetics and other characteristics of the humoral re- 
sponse differ considerably depending on whether the hu- 
moral response results from activation of naive lymphocytes 
(primary response) or memory lymphocytes (secondary re- 
sponse). In both cases, activation leads to production of se- 
creted antibodies of various isotypes, which differ in their 
ability to mediate specific effector functions (see Table 4-2). 

Thefirst contact of an exogenous antigen with an individ- 
ual generates a primary humoral response, characterized by 
theproduction of antibody- secreting plasma cellsand mem- 
ory B cells. As Chapter 3 showed, the kinetics of the primary 
response, as measured by serum antibody level, depend on 
the nature of the antigen, the route of antigen administra- 
tion, the presence or absence of adjuvants, and thespeciesor 
strain being immunized. 

In all cases, however, a primary response to antigen is 
characterized by a lag phase, during which naive B cells un- 
dergo clonal selection, subsequent clonal expansion, and dif- 



ferentiation into memory cells or plasma cells (Figure 11-14). 
The lag phase isfollowed by a logarithmic increase in serum 
antibody level, which reaches a peak, plateaus for a variable 
time, and then declines. The duration of the lag phase varies 
with the nature of the antigen. I mmunization of mice with 
an antigen such as sheep red blood cells (SRBCs) typically re- 
sults in a lag phase of 3-4 days. Eight or nine successive cell 
divisions of activated B cells during days 4 and 5 then gener- 
ate plasma and memory cells. Peak plasma- cell levels are at- 
tained at day 4-5; peak serum antibody levels are attained by 
around day 7-10. For soluble protein antigens, the lag phase 
is a little longer, often lasting about a week, peak plasma-cell 
levels are attained by 9-10 days, and peak serum titers are 
present by around 14 days. During a primary humoral re- 
sponse, I gM is secreted initially, often followed by a switch to 
an increasing proportion of IgG. Depending on the persis- 
tence of the antigen, a primary response can last for various 
periods, from only a few days to several weeks. 

The memory B cells formed during a primary response 
stop dividing and enter the G phase of the cell cycle. These 
cells have variable lifespans, with some persisting for the life 
of the individual. The capacity to develop a secondary hu- 
moral response (see Figure 11-14) depends on the existence 
of this population of memory B cells as well as memory 
T cells. Activation of memory cells by antigen resu Its in a sec- 
ondary antibody response that can bedistinguished from the 
primary response in several ways (Table 11-4). The secon- 
dary response has a shorter lag period, reaches a greater mag- 



VI SUALIZI N G CONCEPTS 






5 o.i 







Total -. 


Primary 
response 


->*■ 


/ Secondary 
/ response 


Total v 




/ igG 


- IgM ^/^\ \ 


w^IgG 


/ 


<-lag-V 




//igM \. 



T 

l°Ag 



T 

2°Ag 
Time after immunization 



Concentration and isotype of serum antibody scale. The time units are not specified because the kinetics differ 
following primary (1°) and secondary (2°) immunization with anti- somewhat with type of antigen, administration route presence or 
gen. The antibody concentrations are plotted on a logarithmic absence of adjuvant, and the species or strain of animal. 



B-Cell Generation, Activation, and Differentiator 



TABLE 11-4 



Property 



."<■ >^W 



Secondary response 



3 



Responding B cell 

Lag period following antigen 

administration 
Time of peak response 
M agnitude of peak antibody 

response 
Isotype produced 
Antigens 

Antibody affinity 



Naive (virgin) B cell 
Generally 4-7 days 

7-10 days 

Varies depending on antigen 

IgM predominates early in the response 
Thymus-dependent and thymus- 

independent 
Lower 



Memory B cell 
Generally 1-3 days 

3-5 days 

Generally 100-1000 times higher 

than primary response 
IgG predominates 
Thymus-dependent 

Higher 



nitude, and lasts longer. The secondary response is also char- 
acterized by secretion of antibody with a higher affinity for 
theantigen,and isotypes other than IgM predominate. 

A major factor in the more rapid onset and greater mag- 
nitude of secondary responses is the fact that the population 
of memory B cells specific for a given antigen is considerably 
larger than the population of corresponding naive B cells. 
Furthermore, memory cells are more easily activated than 
naive B cells. The processes of affinity maturation and class 
switching are responsible for the higher affinity and different 
isotypes exhibited in a secondary response. The higher levels 
of antibody coupled with the overall higher affinity provide 
an effective host defense against reinfection. The change in 
iso typeprovides an ti bodies whose effector functionsarepar- 
ticularly suited to a given pathogen. 

The existence of long-lived memory B eel Is accounts for a 
phenomenon called "original antigenic sin," which was first 
observed when the antibody response to influenza vaccines 
was monitored in adults. Monitoring revealed that immu- 
nization with an influenza vaccine of one strain elicited an 
antibody response to that strain but, paradoxically, also 
elicited an antibody response of greater magnitude to an- 
other influenza strain that the individual had been exposed 
to during childhood. It was as if the memory of the first anti- 
gen exposurehad left a life-long imprint on theimmunesys- 
tem. This phenomenon can be explained by the presence of a 
memory-cell population, elicited by the influenza strain en- 
countered in childhood, that is activated by cross- reacting 
epitopeson thevaccinestrain encountered later.Thisprocess 
then generates a secondary response, characterized by anti- 
bodies with higher affinity for the earlier viral strain. 

T H elper Cells Play a Critical Role in the 
Humoral Response to Hapten-Carrier 
Conjugates 

As Chapter 3 described, when animals are immunized with 
small organic compounds (haptens) conjugated with large 



proteins (carriers), theconjugateinducesa humoral immune 
response consisting of antibodies both to hapten epitopes 
and to unaltered epitopes on the carrier protein. Hapten- 
carrier conjugates provided immunologistswith an ideal sys- 
tem for studying cellular interactions of the humoral re- 
sponse, and such studies demonstrated that the generation 
|of a humoral antibody response requires recognition of 
theantigenbybothT H cellsand B cells, e